The Open Dentistry Journal




ISSN: 1874-2106 ― Volume 13, 2019
CLINICAL TRIAL STUDY

Antibacterial Activity of Phytochemical Extracts and Endophytic Fungi of Carapa Guianensis Against Enterococcus Faecalis in Endodontic Infections An In Vitro Study



Maria do Carmo M. de Miranda1, Clarice M. Carvalho2, Fernando S.E.D.V. Faria2, Antonio M-S. Nobreza3, Thiago M. Pereira3, Keliin Pivatto3, Marcus V.C. da Costa4, Orlando A. Guedes3, Cyntia R. de Araújo Estrela3, Álvaro H. Borges3, *
1 Institute of Biodiversity and Biotechnology of the Legal Amazon, Federal University of Acre, Rio Branco, Acre, Brazil
2 Center for Biological and Nature Sciences, Federal University of Acre, Rio Branco, Acre, Brazil
3 Department of Oral Sciences, University of Cuiaba, Cuiaba, Mato Grosso, Brazil
4 Faculty of Medicine, Federal University of Goias, Goiânia, Goiás, Brazil

Abstract

Objective:

The objective of this study was to evaluate the antibacterial activity of phytochemical extracts and endophytic fungi of Carapa guianensis against Enterococcus faecalis. Carapa guianensis leaves and stems were collected to obtain phytochemical extracts and fungal metabolites and evaluated for in vitro antibacterial activity against E. faecalis using the disc diffusion method and dentin blocks with bacterial biofilm.

Methods:

Thirty dentin blocks were prepared and contaminated for 60 days with E. faecalis. The specimens were randomly divided into 6 experimental groups according to the test solution used: G1 – hexane stem extract of Carapa guianensis; G2 – methanol stem extract of Carapa guianensis; G3 – methanol leaf extract of Carapa guianensis; G4 – ethyl acetate extract of the endophytic fungus Penicillium isolated from Carapa guianensis; G5 – negative control, with no addition of bacterial inoculum; G6 – positive control.

Results:

Bacterial growth was analyzed by spectrophotometry after 14 days of direct contact between the extracts and dentin blocks. The hexane-stem, methanol-stem, methanol-leaf, and ethyl-acetate endophytic fungus Penicillium extracts inhibited bacterial growth in 100% of the samples.

Conclusion:

The present study demonstrated the antibacterial potential of phytochemical extracts and endophytic fungi of Carapa guianensis against E. faecalis.

Keywords:: Endodontics. Enterococcus faecalis. Phytochemicals, Carapa guianensis, Endophytic fungi, Penicillium.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 249
Last Page: 254
Publisher Id: TODENTJ-13-249
DOI: 10.2174/1874210601913010249

Article History:

Received Date: 25/03/2019
Revision Received Date: 27/06/2019
Acceptance Date: 01/07/2019
Electronic publication date: 31/07/2019
Collection year: 2019

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© 2019 De Miranda et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Oral Sciences, School of Dentistry, University of Cuiabá, Avenida Manoel José de Arruda, 3100, Jardim Europa, 78065-900, Cuiabá, MT, Brazil; Tel: +55 65 3363-1264;
E-mail: alvarohborges@gmail.com





1. INTRODUCTION

The success of endodontic treatment is related to the elimination of microorganisms from the root canal system [1Zancan RF, Canali LCF, Tartari T, Andrade FB, Vivan RR, Duarte MAH. Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research? Braz Oral Res 2018; 32e46[http://dx.doi.org/10.1590/1807-3107bor-2018.vol32.0046] [PMID: 2 9846391] , 2Estrela C, Costa E Silva R, Urban RC, et al. Demetallization of Enterococcus faecalis biofilm: A preliminary study. J Appl Oral Sci 2018; 26e20170374[http://dx.doi.org/10.1590/1678-7757-2017-0374] [PMID: 29451651] ]. Persistent infection after endodontic treatment, resulting from remnants of bacteria in root canal systems in areas of anatomical complexity and difficult access, continues to present a challenge in endodontics [1Zancan RF, Canali LCF, Tartari T, Andrade FB, Vivan RR, Duarte MAH. Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research? Braz Oral Res 2018; 32e46[http://dx.doi.org/10.1590/1807-3107bor-2018.vol32.0046] [PMID: 2 9846391] ]. Thus, recurrent periapical infections are mainly associated with Enterococcus faecalis [2Estrela C, Costa E Silva R, Urban RC, et al. Demetallization of Enterococcus faecalis biofilm: A preliminary study. J Appl Oral Sci 2018; 26e20170374[http://dx.doi.org/10.1590/1678-7757-2017-0374] [PMID: 29451651] ]. Some factors allow E. faecalis to survive in root canal systems for long periods, such as resistance to antimicrobials, ability to survive for long periods without nutrients, capability to adhere to dentin, invasion of dentinal tubules, and ability to bind to collagen and form a biofilm [3Dalmia S, Gaikwad A, Samuel R, Aher G, Gulve M, Kolhe S. Antimicrobial efficacy of different endodontic sealers against Enterococcus faecalis: An In vitro study. J Int Soc Prev Community Dent 2018; 8(2): 104-9.[http://dx.doi.org/10.4103/jispcd.JISPCD_29_18] [PMID: 29780734] ].

Several intracanal medicaments have been proposed for the treatment of endodontic infections, of which calcium hydroxide is the most important due to its antimicrobial and anti-inflammatory properties [4Saatchi M, Shokraneh A, Navaei H, Maracy MR, Shojaei H. Anti- bacterial effect of calcium hydroxide combined with chlorhexidine on Enterococcus faecalis: A systematic review and meta-analysis. J Appl Oral Sci 2014; 22(5): 356-65.[http://dx.doi.org/10.1590/1678-775720140032] [PMID: 25466470] ]. Associated with induction of mineralized tissue formation, dissolution of organic matter, and biocompatibility, calcium hydroxide acts on the cell walls of bacteria, causing damage to the bacterial cytoplasmic membrane, protein denaturation, and DNA damage [4Saatchi M, Shokraneh A, Navaei H, Maracy MR, Shojaei H. Anti- bacterial effect of calcium hydroxide combined with chlorhexidine on Enterococcus faecalis: A systematic review and meta-analysis. J Appl Oral Sci 2014; 22(5): 356-65.[http://dx.doi.org/10.1590/1678-775720140032] [PMID: 25466470] ]. However, several studies have indicated that calcium hydroxide is ineffective in inhibiting the growth of E. faecalis, because the buffering effect of dentin neutralizes the action of this medicament at deeper layers of dentinal tubules, resulting in the survival of this microorganism [5Cwikla SJ, Bélanger M, Giguère S, Progulske-Fox A, Vertucci FJ. Dentinal tubule disinfection using three calcium hydroxide formulations. J Endod 2005; 31(1): 50-2.[http://dx.doi.org/10.1097/01.DON.0000134291.03828.D1] [PMID: 1 5614007] ].

Biological activity of some plant species has been raising interest in research due to the production of bioactive substances [6de Rezende GP, da Costa LR, Pimenta FC, Baroni DA. In vitro antimicrobial activity of endodontic pastes with propolis extracts and calcium hydroxide: A preliminary study. Braz Dent J 2008; 19(4): 301-5.[http://dx.doi.org/10.1590/S0103-64402008000400003] [PMID: 1918 0318] -9Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645] ]. Carapa guianensis Aublet, known as Andiroba, presents applications in Brazilian medicine and in other countries of the Amazonian forest [7Penido C, Conte FP, Chagas MSS, Rodrigues CAB, Pereira JF, Henriques MG. Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice. Inflamm Res 2006; 55(11): 457-64.[http://dx.doi.org/10.1007/s00011-006-5161-8] [PMID: 17122962] ]. Studies evaluating its biological activity have reported antimicrobial activity, cicatrization, anti-inflammatory [7Penido C, Conte FP, Chagas MSS, Rodrigues CAB, Pereira JF, Henriques MG. Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice. Inflamm Res 2006; 55(11): 457-64.[http://dx.doi.org/10.1007/s00011-006-5161-8] [PMID: 17122962] -9Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645] ]. However, there is no report in the literature about its use in the endodontic therapy.

Endotoxic microorganisms, which inhabit the interior of the plant, without causing any damage, produce secondary metabolites that also generate bioactive principles [7Penido C, Conte FP, Chagas MSS, Rodrigues CAB, Pereira JF, Henriques MG. Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice. Inflamm Res 2006; 55(11): 457-64.[http://dx.doi.org/10.1007/s00011-006-5161-8] [PMID: 17122962] -9Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645] ]. Endophytic fungi are important and can produce toxins, antibiotics, drugs, growth factors and many products of biotechnological interest, being also a natural source of new drugs that can control microorganisms that cause infections [7Penido C, Conte FP, Chagas MSS, Rodrigues CAB, Pereira JF, Henriques MG. Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice. Inflamm Res 2006; 55(11): 457-64.[http://dx.doi.org/10.1007/s00011-006-5161-8] [PMID: 17122962] -9Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645] ]. Therefore, the purpose of this in vitro study was to evaluate the efficacy of phytochemical extracts and endophytic fungi of Carapa guianensis against E. faecalis biofilm on dentin.

2. MATERIALS AND METHODS

2.1. Production of Phytochemical Extracts From Carapa Guianensis

The leaves and stems were washed with tap water, dried at room temperature and grounded to a fine powder using an electric mill. Extracts were obtained according to the technique described by Celeghini et al. (2001) [10Celeghini RMS, Vilegas JHY, Lanças FM. Extraction and quantitative HPLC analysis of coumarin in hydroalcoholic extracts of Mikania glomercita Spreng (guaco) leaves. J Braz Chem Soc 2001; 12(6): 706-9.[http://dx.doi.org/10.1590/S0103-50532001000600003] ], where 100 g of powered plant material was extracted with hexane and methanol (100 g of samples for 400 mL of solvent) under sonication for 20 minutes.

2.2. Production of Endophytic Fungal Extracts From Carapa Guianensis

The Carapa guianensis endophytic fungus no. 2477, identified as Penicillium sp., was grown on a larger scale for metabolite extraction. This endophytic fungus was cultured in 30 Petri dishes (80 × 15 mm) containing potato dextrose agar medium for 14 days at 28 °C. Five plates served as an inoculum for each 1000-mL Erlenmeyer flask containing 500 mL of potato dextrose broth. Six Erlenmeyer flasks were inoculated and incubated without shaking for 14 days at 28 °C. The fungal mycelium was separated from the liquid medium by filtration using filter paper. The filtrate was extracted 3 times by liquid-liquid extraction with ethyl acetate and concentrated in a rotary evaporator under reduced pressure in a water bath at 40 °C.

2.3. Bioassay of In Vitro Antibacterial Activity

The phytochemical and endophytic fungus Penicillium extracts were dissolved in Dimethyl Sulfoxide (DMSO) and analyzed at the initial concentration of 1 g mL-1 using the disc diffusion test with E. faecalis (ATCC 4083) as the test microorganism. To prepare the inoculum, three well-isolated bacterial colonies of the same morphological type were selected from a Mueller-Hinton agar plate culture. The top of each colony was touched with a loop, and the growth was transferred into a tube containing 3 mL of Luria-Bertani broth and incubated at 37 °C for 4 hours. The turbidity of the actively growing broth culture was adjusted with 0.9% saline to obtain turbidity optically comparable to that of the 0.5 McFarland standard, resulting in a suspension containing approximately 1.5 × 108 Colony-Forming Units (CFU) per mL.

After adjusting the turbidity, a sterile cotton swab was dipped into the suspension and inoculated into Mueller-Hinton agar. The procedure was repeated 2 more times, rotating the plate approximately 60° each time to ensure an even distribution of the inoculum, and the rim of the Petri dish was swabbed as a final step. After inoculation, the lid of the plate was left ajar for 5 minutes to allow for any excess surface moisture to be absorbed before applying the paper discs.

Sterile paper discs were dispensed onto the surface of the inoculated agar plate, and 20 μL of the extracts under analysis were placed onto the discs. The plates were stored for 2 hours at 4 °C to allow diffusion of the analyzed samples and subsequently incubated at 37 °C for 24 hours. After the 24-hour incubation, each plate was examined for the presence of an inhibition halo, which was measured in millimeters using a digital caliper. DMSO served as a negative control. All antibacterial activity analyses were performed in triplicate. The extracts with positive antagonism were dissolved in DMSO and analyzed at concentrations of 1g mL-1, 500 mg mL-1, 250 mg mL-1, 125 mg mL-1, 62.5 mg mL-1, and 31.25 mg mL-1 for the Minimum Inhibitory Concentration (MIC).

2.4. Preparation of Dentin Blocks

Thirty dentin blocks were prepared from extracted single-rooted human teeth. All teeth used in the study had an indication for extraction for periodontal or prosthetic reasons. Teeth with root canal obliteration, root dilaceration, or root canal filling were excluded. The teeth were sectioned to obtain dentin blocks of 4 mm height and 4 mm length. Two layers of nail polish were applied to the external surfaces of all dentin blocks to prevent infiltration. The specimens were washed twice under sonication with 17% EDTA for 5 minutes and then rinsed with sterile distilled water under sonication for 10 minutes. The dentin blocks were autoclaved at 120 °C for 30 minutes and subsequently incubated in 7 mL of Brain Heart Infusion (BHI) broth at 37 °C for 48 hours to ensure sterilization, so that, after this incubation period, no bacterial growth was observed.

2.5. Contamination of Dentin Blocks

For contamination, E. faecalis (ATCC 29212) was inoculated into 7 mL of BHI broth, incubated at 37 °C for 24 hours and subsequently inoculated onto the surface of the BHI agar plate under the same incubation conditions. Microbial cells were suspended in saline solution to a concentration of 3 x 108 cells mL-1, matched to 1 McFarland standard.

The dentin blocks were inoculated with 0.01 mL of the bacterial inoculum using a pure culture of E. faecalis grown for 24 hours and adjusted to 1 McFarland standard. The contaminated blocks were incubated for 60 days at 37 °C in a humid chamber. Five uncontaminated specimens (negative control group) were incubated at 37 °C during the contamination period to monitor sample sterility, while the positive control group consisted of 5 contaminated specimens that were incubated at 37 °C throughout the experimental period to determine the viability of the biological indicator. After 60 days, under aseptic conditions, each dentin block was removed from the inoculated suspension and rinsed with 5 mL of sterile distilled water to remove the culture and non-adherent cells. Bacterial growth was determined by turbidity of the culture medium.

2.6. Application of Phytochemical Extracts and Endophytic Fungi of Carapa guianensis to Dentin Blocks

The selected extracts were distributed in 30 Eppendorf tubes for each experimental group taking into account the MIC previously determined in the disc diffusion test. The contaminated dentin blocks were randomly selected and immersed in the test solutions, accounting for 6 experimental groups, for 14 days, as described in Table 1, and an initial spectrophotometer reading was taken. After 14 days, the dentin blocks were removed from the experimental solutions, rinsed with 5 mL of sterile distilled water, transferred individually to 7 mL of BHI broth, with the addition of two neutralizers (sodium thiosulfate and Tween 80), and incubated at 37 °C for 48 hours.

2.7. Microbiological Analysis

The tubes containing dentin blocks were incubated at 37°C for 48 hours, and microbial growth was analyzed. Visual readings were taken over the 48-hour period for the absence (negative) or presence (positive) of microbial growth in the 6 groups, as determined by turbidity of the culture medium. After 48 hours, 0.1 mL of BHI medium was inoculated into 20 tubes containing 5 mL of Letheen broth, which were then incubated under the same conditions for an additional 48 hours, when a spectrophotometer reading was taken. Microbial concentration was analyzed using a UV spectrophotometer (Model NI- 1600UV; Nova Instruments) adjusted for reading at a wavelength of 600 nm.

2.8. Statistical Analysis

Distribution of random errors around the mean (normality) and presence or absence of homogenous variances were tested, respectively, by the Shapiro-Wilk and Lévene tests. Sequentially, Student's T-test was used to compare the data between the means obtained in the initial and final analyzes. Level of significance was set at 5%.

3. RESULTS

The results of the disc diffusion test indicated that the methanol-leaf, hexane-stem, and methanol-stem extracts and the ethyl-acetate endophytic fungus Penicillium extract showed antibacterial activity against E. faecalis (Table 2). The MICs of the phytochemical and ethylacetate endophytic fungus Penicillium extracts that showed activity against E. faecalis are shown in Table 2.

Table 1
Experimental groups for antibacterial analysis of Carapa guianensis endophytic fungi against Enterococcus faecalis biofilm.



Table 2
Evaluation of minimum inhibitory concentration of the phytochemical extracts and endophytic fungi of Carapa guianensis against to Enterococcus faecalis (ATCC 29212).



Table 3
Evaluation of bacterial growth after the application of phytochemical extracts and endophytic fungi of Carapa guianensis against to Enterococcus faecalis biofilm.


Regarding the action of the phytochemical extracts and endophytic fungi of Carapa guianensis against contamination of dentin blocks with E. faecalis, showed that all extracts in the experimental groups 1, 2, 3, and 4 had antibacterial effects presenting the elimination of E. faecalis grown on dentin blocks (p<0.05) (Table 3). In group 5 (negative control), no bacterial growth was observed (p>0.05), confirming the sterility of the culture medium. In group 6 (positive control), the viability of the strains used in the experiment was confirmed.

4. DISCUSSION

The viability of the remaining microorganisms after instrumentation and disinfection of root canals contributes significantly to endodontic treatment failure [1Zancan RF, Canali LCF, Tartari T, Andrade FB, Vivan RR, Duarte MAH. Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research? Braz Oral Res 2018; 32e46[http://dx.doi.org/10.1590/1807-3107bor-2018.vol32.0046] [PMID: 2 9846391] ]. In the absence of clinical evidence that intracanal medicaments are sufficiently effective against persistent bacterial infections, there is a need for further investigation of new medicaments for use in periradicular conditions [2Estrela C, Costa E Silva R, Urban RC, et al. Demetallization of Enterococcus faecalis biofilm: A preliminary study. J Appl Oral Sci 2018; 26e20170374[http://dx.doi.org/10.1590/1678-7757-2017-0374] [PMID: 29451651] ].

The hexane-stem, methanol-stem, methanol-leaf, and ethyl-acetate endophytic fungus Penicillium sp. extracts of Carapa guianensis analyzed in the present study showed antibacterial activity against E. faecalis in disc diffusion and dental biofilm tests. Several methods have been proposed to evaluate the antibacterial potential of medicaments used in the treatment of endodontic infections, such as disc diffusion method [11Zare Jahromi M, Toubayani H, Rezaei M. Propolis: A new alternative for root canal disinfection. Iran Endod J 2012; 7(3): 127-33.[PMID: 23056131] ], dentin infection and disinfection test [12Farhad AR, Barekatain B, Allameh M, Narimani T. Evaluation of the antibacterial effect of calcium hydroxide in combination with three different vehicles: An in vitro study. Dent Res J (Isfahan) 2012; 9(2): 167-72.[http://dx.doi.org/10.4103/1735-3327.95231] [PMID: 22623933] ], and dentin block biofilm models [13Estrela C, Sydney GB, Figueiredo JA, Estrela CR. A model system to study antimicrobial strategies in endodontic biofilms. J Appl Oral Sci 2009; 17(2): 87-91.[http://dx.doi.org/10.1590/S1678-77572009000200003] [PMID: 1927 4391] ]. All these methods allow sufficient time for satisfactory colonization of bacterial species with virulence and adherence properties [11Zare Jahromi M, Toubayani H, Rezaei M. Propolis: A new alternative for root canal disinfection. Iran Endod J 2012; 7(3): 127-33.[PMID: 23056131] -13Estrela C, Sydney GB, Figueiredo JA, Estrela CR. A model system to study antimicrobial strategies in endodontic biofilms. J Appl Oral Sci 2009; 17(2): 87-91.[http://dx.doi.org/10.1590/S1678-77572009000200003] [PMID: 1927 4391] ]. Additionally, Estrela et al. (2009) [13Estrela C, Sydney GB, Figueiredo JA, Estrela CR. A model system to study antimicrobial strategies in endodontic biofilms. J Appl Oral Sci 2009; 17(2): 87-91.[http://dx.doi.org/10.1590/S1678-77572009000200003] [PMID: 1927 4391] ] regarding colonization in dentin blocks validated the methodology presented in this study concluding that this biofilm model is viable for studies on antimicrobial strategies and allows satisfactory colonization of the selected bacterial species with virulence and adherence properties.

The biotechnological potential of several plant species against microorganisms present in root canals has been reported in the literature [9Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645] -13Estrela C, Sydney GB, Figueiredo JA, Estrela CR. A model system to study antimicrobial strategies in endodontic biofilms. J Appl Oral Sci 2009; 17(2): 87-91.[http://dx.doi.org/10.1590/S1678-77572009000200003] [PMID: 1927 4391] ]. However, there are currently no reports on the biological activity of extracts obtained from the leaves, stems or endophytic fungi of Carapa guianensis against E. faecalis. Some studies were found that analyzed the antibacterial activity of phytochemical extracts against E. faecalis, such as the study conducted by Costa et al. (2010) [14Costa COD, Ribeiro PR, Castro RD, Fernandez LG. Evaluation of antioxidant activity in commercial samples of Schinus terebinthifolius (aroeira vermelha). Rev Ciênc Méd Biol 2010; 12(3): 312-7.[http://dx.doi.org/10.9771/cmbio.v12i3.7093] ], who found that ethanol extracts of the plant species Schinus terebinthifolius, Astronium urundeuva, Ximenia americana, and Syderoxylum obtusifolium showed antimicrobial activity. Nath et al. (2012) [15Nath A, Raghunatha P, Joshi SR. Diversity and biological activities of endophytic fungi of Emblica officinalis, An ethnomedicinal plant of India. Mycobiology 2012; 40(1): 8-13.[http://dx.doi.org/10.5941/MYCO.2012.40.1.008] [PMID: 22783128] ] in an attempt to evaluate the antimicrobial activity of fungal endophytes inhabiting Emblica officinalis assessed their effect against four bacteria namely, Escherichia coli, Enteroccocus faecalis, Salmonella enterica ser. paratyphi and Streptococcus pyogenes, and the fungus Candida albicans. In general, the fungal extracts inhibited the growth of all tested organisms except Escherichia coli.

Fungi of the genus Penicillium can produce several secondary metabolites, and 132 different metabolites have been reported to be produced by fungi of this genus, including pigments and antimicrobial compounds [16Frisvad J, Samson RA. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol 2004; 49: 1-174.]. Penicillium sp. are characterized by the abundant production of spores and have been widely investigated in the search for bioactive compounds [17Takahashi JA, Esther MFL. Occurrence and structural diversity of fungal metabolites with antibiotic activity. Quim Nova 2008; 31(7): 1807-13.[http://dx.doi.org/10.1590/S0100-40422008000700036] ], with endophytic fungal strains showing neuroprotective [18Wu Y, Qiao F, Xu G, et al. Neuroprotective metabolites from the endophytic fungus Penicillium citrinum of the mangrove Bruguiera gymnorrhiza. Phytochem Lett 2015; 12: 148-52.[http://dx.doi.org/10.1016/j.phytol.2015.03.007] ], antitumor [19Zheng C, Chen Y, Jiang L, Shi X. Antiproliferative metabolites from the endophytic fungus Penicillium sp. FJ-1 isolated from a mangrove Avicennia marina. Phytochem Lett 2014; 10: 272-5.[http://dx.doi.org/10.1016/j.phytol.2014.10.011] ], and antibacterial [20Jouda JB, Kusari S, Lamshöft M, et al. Penialidins A-C with strong antibacterial activities from Penicillium sp., An endophytic fungus harboring leaves of Garcinia nobilis. Fitoterapia 2014; 98: 209-14.[http://dx.doi.org/10.1016/j.fitote.2014.08.011] [PMID: 25128427] ] activities. In this study the endophytic fungus Penicillium sp demonstrated promising results corroborating the idea of its application against E. faecalis in order to overcome deficiencies of the current intracanal medications.

Studies have demonstrated the antimicrobial activity of Carapa guianensis against several microorganisms [8Nayak BS, Kanhai J, Milne DM, et al. Investigation of the wound healing activity of Carapa guianensis L. (Meliaceae) bark extract in rats using excision, incision, and dead space wound models. J Med Food 2010; 13(5): 1141-6.[http://dx.doi.org/10.1089/jmf.2009.0214] [PMID: 20828307] , 9Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645] , 21Shu-Hua QI, Da-Gang WU, Yun-Bao MA, Xiao-Dang L. A novel flavane from Carapa guianensis. Acta Bot Sin 2003; 45(9): 1129-33., 22Costa-Silva JH, Lyra MMA, Lima CR, et al. A toxicological evaluation of the effect of Carapa guianensisAublet on pregnancy in Wistar rats. J Ethnopharmacol 2007; 112(1): 122-6.[http://dx.doi.org/10.1016/j.jep.2007.02.004] [PMID: 17368776] ], but none of them investigated E. faecalis, thereby hindering a proper comparison with the results obtained in the present study. Costa-Silva et al., (2007) [22Costa-Silva JH, Lyra MMA, Lima CR, et al. A toxicological evaluation of the effect of Carapa guianensisAublet on pregnancy in Wistar rats. J Ethnopharmacol 2007; 112(1): 122-6.[http://dx.doi.org/10.1016/j.jep.2007.02.004] [PMID: 17368776] ] found that the use of Carapa guianensis seed oil in pregnant rats produced no toxic effect. Miranda-Junior et al., (2012) [23Miranda Júnior RN, Dolabela MF, da Silva MN, Póvoa MM, Maia JG. Antiplasmodial activity of the andiroba (Carapa guianensis Aubl., Meliaceae) oil and its limonoid-rich fraction. J Ethnopharmacol 2012; 142(3): 679-83.[http://dx.doi.org/10.1016/j.jep.2012.05.037] [PMID: 22659195] ] evaluated the antispasmodic effects and toxicity activity of Carapa guianensis in mice, and no toxic limonoids were identified. Nevertheless, studies evaluating the antimicrobial actions of Carapa guianensis phytochemical compounds still show promising perspectives [24Oliveira IDSDS, Moragas Tellis CJ, Chagas MDSDS, et al. Carapa guianensis Aublet (Andiroba) seed oil: Chemical composition and antileishmanial activity of limonoid-rich fractions. BioMed Res Int 2018; 20185032816[http://dx.doi.org/10.1155/2018/5032816] [PMID: 30258850] ,25Pereira TB, Rocha E Silva LF, Amorim RC, et al. In vitro and in vivo anti-malarial activity of limonoids isolated from the residual seed biomass from Carapa guianensis (andiroba) oil production. Malar J 2014; 13: 317.[http://dx.doi.org/10.1186/1475-2875-13-317] [PMID: 25124944] ]. Carapa guianensis demonstrated effects against protozoa of the genera Plasmodium and Trypanosoma, due to the limonoids that exist in its compound [25Pereira TB, Rocha E Silva LF, Amorim RC, et al. In vitro and in vivo anti-malarial activity of limonoids isolated from the residual seed biomass from Carapa guianensis (andiroba) oil production. Malar J 2014; 13: 317.[http://dx.doi.org/10.1186/1475-2875-13-317] [PMID: 25124944] ]. As the chemical constituents of the Carapa guianensis exhibited anti-Leishmania activity [24Oliveira IDSDS, Moragas Tellis CJ, Chagas MDSDS, et al. Carapa guianensis Aublet (Andiroba) seed oil: Chemical composition and antileishmanial activity of limonoid-rich fractions. BioMed Res Int 2018; 20185032816[http://dx.doi.org/10.1155/2018/5032816] [PMID: 30258850] ] due to the limonoids 11β-hydroxygedunin and 6α,11 β-diacetoxygedunin, identified in the active limonoid-rich fractions [24Oliveira IDSDS, Moragas Tellis CJ, Chagas MDSDS, et al. Carapa guianensis Aublet (Andiroba) seed oil: Chemical composition and antileishmanial activity of limonoid-rich fractions. BioMed Res Int 2018; 20185032816[http://dx.doi.org/10.1155/2018/5032816] [PMID: 30258850] ].

Only one study was found considering the endophytic fungal community associated with Carapa guianensis as well as its potential to produce bioactive compounds. In this study, a total of 162 fungi were isolated from the leaves of Carapa guianensis, and some of these fungi showed biological activity, such as Colletotrichum sp. and Pilidiella wangiensis, which showed antibacterial activity; however, the activity against E. faecalis was not evaluated [26Ferreira MC, Vieira MDLA, Zani CL, et al. Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae) Biochem Syst Ecol 2015; 59: 36-44.[http://dx.doi.org/10.1016/j.bse.2014.12.017] ]. Most bioactive substances obtained from fungal extracts can be used directly as medications or even modified to obtain a new molecule with different properties, such as increased activity, selective toxicity, and reduced undesirable side effects [16Frisvad J, Samson RA. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol 2004; 49: 1-174.-26Ferreira MC, Vieira MDLA, Zani CL, et al. Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae) Biochem Syst Ecol 2015; 59: 36-44.[http://dx.doi.org/10.1016/j.bse.2014.12.017] ]. Even when these substances do not yield the expected activity, they may serve as prototypes for the planning and development of new molecules, which further highlight the importance of studying endophytic fungi as a source of new substances against recurrent endodontic infections [16Frisvad J, Samson RA. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol 2004; 49: 1-174.-26Ferreira MC, Vieira MDLA, Zani CL, et al. Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae) Biochem Syst Ecol 2015; 59: 36-44.[http://dx.doi.org/10.1016/j.bse.2014.12.017] ]. Substances tested against Enterococcus faecalis demonstrated promising in vitro results. New studies should be designed to evaluate other biological properties of these compounds.

CONCLUSION

The present study demonstrated the antibacterial potential of phytochemical extracts and endophytic fungi of Carapa guianensis against E. faecalis. Nevertheless, further antimicrobial, biocompatibility, and toxicity assays of the active compounds are warranted to confirm these findings.

ETHICS APPROVAL AND CONSENT TO PARTI-CIPATE

The study was approved by the Human Research Ehtics Committee of Fedral University of Goiás- number 1.214.

HUMAN AND ANIMAL RIGHTS

No animals were used in this research. All research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013 (http://ethics.iit. edu/ecodes/node/3931).

CONSENT FOR PUBLICATION

The participants provided written informed consent to be involved in this study.

AVAILABILITY OF DATA AND MATERIALS:

The data supporting the findings of the article is available in the [Federal University of Acre] at [https://bionorte.org.br/ programa-de-pos-graduacao/corpo-discente.htm?idp=1&pgn =6].

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

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[9] Santos RCV, dos Santos Alves CF, Schneider T, et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. J Invertebr Pathol 2012; 109(3): 265-8.[http://dx.doi.org/10.1016/j.jip.2011.12.002] [PMID: 22200645]
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[14] Costa COD, Ribeiro PR, Castro RD, Fernandez LG. Evaluation of antioxidant activity in commercial samples of Schinus terebinthifolius (aroeira vermelha). Rev Ciênc Méd Biol 2010; 12(3): 312-7.[http://dx.doi.org/10.9771/cmbio.v12i3.7093]
[15] Nath A, Raghunatha P, Joshi SR. Diversity and biological activities of endophytic fungi of Emblica officinalis, An ethnomedicinal plant of India. Mycobiology 2012; 40(1): 8-13.[http://dx.doi.org/10.5941/MYCO.2012.40.1.008] [PMID: 22783128]
[16] Frisvad J, Samson RA. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol 2004; 49: 1-174.
[17] Takahashi JA, Esther MFL. Occurrence and structural diversity of fungal metabolites with antibiotic activity. Quim Nova 2008; 31(7): 1807-13.[http://dx.doi.org/10.1590/S0100-40422008000700036]
[18] Wu Y, Qiao F, Xu G, et al. Neuroprotective metabolites from the endophytic fungus Penicillium citrinum of the mangrove Bruguiera gymnorrhiza. Phytochem Lett 2015; 12: 148-52.[http://dx.doi.org/10.1016/j.phytol.2015.03.007]
[19] Zheng C, Chen Y, Jiang L, Shi X. Antiproliferative metabolites from the endophytic fungus Penicillium sp. FJ-1 isolated from a mangrove Avicennia marina. Phytochem Lett 2014; 10: 272-5.[http://dx.doi.org/10.1016/j.phytol.2014.10.011]
[20] Jouda JB, Kusari S, Lamshöft M, et al. Penialidins A-C with strong antibacterial activities from Penicillium sp., An endophytic fungus harboring leaves of Garcinia nobilis. Fitoterapia 2014; 98: 209-14.[http://dx.doi.org/10.1016/j.fitote.2014.08.011] [PMID: 25128427]
[21] Shu-Hua QI, Da-Gang WU, Yun-Bao MA, Xiao-Dang L. A novel flavane from Carapa guianensis. Acta Bot Sin 2003; 45(9): 1129-33.
[22] Costa-Silva JH, Lyra MMA, Lima CR, et al. A toxicological evaluation of the effect of Carapa guianensisAublet on pregnancy in Wistar rats. J Ethnopharmacol 2007; 112(1): 122-6.[http://dx.doi.org/10.1016/j.jep.2007.02.004] [PMID: 17368776]
[23] Miranda Júnior RN, Dolabela MF, da Silva MN, Póvoa MM, Maia JG. Antiplasmodial activity of the andiroba (Carapa guianensis Aubl., Meliaceae) oil and its limonoid-rich fraction. J Ethnopharmacol 2012; 142(3): 679-83.[http://dx.doi.org/10.1016/j.jep.2012.05.037] [PMID: 22659195]
[24] Oliveira IDSDS, Moragas Tellis CJ, Chagas MDSDS, et al. Carapa guianensis Aublet (Andiroba) seed oil: Chemical composition and antileishmanial activity of limonoid-rich fractions. BioMed Res Int 2018; 20185032816[http://dx.doi.org/10.1155/2018/5032816] [PMID: 30258850]
[25] Pereira TB, Rocha E Silva LF, Amorim RC, et al. In vitro and in vivo anti-malarial activity of limonoids isolated from the residual seed biomass from Carapa guianensis (andiroba) oil production. Malar J 2014; 13: 317.[http://dx.doi.org/10.1186/1475-2875-13-317] [PMID: 25124944]
[26] Ferreira MC, Vieira MDLA, Zani CL, et al. Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae) Biochem Syst Ecol 2015; 59: 36-44.[http://dx.doi.org/10.1016/j.bse.2014.12.017]

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