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Biological properties of Schinus terebinthifolia Raddi essential oil

Abstract

Schinus terebinthifolia Raddi green fruits essential oil (EO) was evaluated regarding its phytochemical profile, antimicrobial and cytotoxic activities, and toxicity. Gas chromatography with mass spectrometry was applied to identify its constituents, thereafter the minimum inhibitory concentration, minimum bactericidal and fungicidal concentrations, and its antibiofilm activity were evaluated. The EO cytotoxicity was assessed in tumor and non-tumor human cells, and in vivo toxicity was evaluated in a Galleria mellonella model. The major constituents of S. terebinthifolia EO were alpha-phellandrene and beta-phellandrene. The EO had a weak activity against all strains of Candida albicans (MIC 1000μg/mL) and had no activity against non-albicans strains, bacteria, and C. albicans biofilm. Cytostatic activity against all tumor cell lines was shown. Additionally, cell viability remained at EO concentrations up to 62.5 μg/mL. At 16 mg/mL, 50% hemolysis was observed, and it had low toxicity in vivo. Overall, the S. terebinthifolia EO was characterized by low antimicrobial and antibiofilm activities, with no evidence of toxicity to human tumor and non-tumor cells.

Keywords:
Anacardiaceae; Medicinal plants; Products with antimicrobial action; Toxicity test

INTRODUCTION

he oral cavity has an important complex and diverse microbiota. However, loss of homeostatic balance between microorganism and host may lead to infections that can compromise health locally or systemically, especially when associated with biofilms (Zhang, 2018Zhang Y. Human oral microbiota and its modulation for oral health. Biomed Pharmacother. 2018;99:883-893. DOI: 10.1016/j.biopha.2018.01.146
https://doi.org/10.1016/j.biopha.2018.01...
). It is estimated that 65% of human infections derive from biofilms, including prevalent clinical conditions of the oral cavity (Scott et al., 2007Scott MG, Dullaghan E, Mookherjee N, Glavas N, Waldbrook M, Thompson A, et al. An antiinfective peptide that selectively modulates the innate immune response. Nat Biotechnol. 2007;25(4):465-472.; De La Fuente-Núñez, Reffuveille, Fernández, 2013De la Fuente-Núñez C, Reffuveille F, Fernández L. Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies. Curr Opin Microbiol. 2013;16(5):580-589. DOI: 10.1016/j.mib.2013.06.013
https://doi.org/10.1016/j.mib.2013.06.01...
) such as oral candidiasis primarily caused by Candida genre of fungi (Swidergall, Filler, 2017Swidergall M, Filler SG. Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses. PLoS Pathog. 2017;13(1):e1006056. DOI: 10.1371/journal.ppat.1006056
https://doi.org/10.1371/journal.ppat.100...
) and conditions associated with bacteria, such as periodontal disease (Roberts, Darveau, 2015Roberts RA, Darveau RP. Microbial Protection and Virulence in Periodontal Tissue as a Function of Polymicrobial Communities: Symbiosis and Dysbiosis. Periodontol 2000. 2015;69(1):18-27. DOI: 10.1111/prd.12087
https://doi.org/10.1111/prd.12087...
).

Pathogen resistance combined with a limited arsenal of antimicrobials (Łukaszuk, Krajewska-kułak, Kułak, 2017Łukaszuk C, Krajewska-kułak E, Kułak W. Retrospective observation of drug susceptibility of Candida strains in the years 1999, 2004, and 2015. PeerJ. 2017;5:e3038. DOI: 10.7717/peerj.3038
https://doi.org/10.7717/peerj.3038...
; Wang, Xu, Hsueh, 2016Wang H, Xu YC, Hsueh PR. Epidemiology of candidemia and antifungal susceptibility in invasive Candida species in the Asia-Pacific region. Future Microbiol. 2016;11(11):1461- 1477. DOI: 10.2217/fmb-2016-0099
https://doi.org/10.2217/fmb-2016-0099...
) has propelled the search for new compounds with good antimicrobial action, such as natural products that are sources of secondary metabolites (Newman, Cragg, 2016Newman DJ, Cragg GM. Natural Products as Sources of New Drugs from 1981 to 2014. J Nat Prod. 2016;79(3):629-661. DOI: 10.1021/acs.jnatprod.5b01055
https://doi.org/10.1021/acs.jnatprod.5b0...
). One of these is Schinus terebinthifolia Raddi, a plant in the Anacardiaceae family that is commonly known in Brazil as aroeira-da-praia, aroeira-vermelha, and pimenta-rosa and as Brazilian pepper tree in English (Carvalho et al., 2013Carvalho M, Melo A, Aragão C, Raffin FN, Moura TFAL. Schinus terebinthifolius Raddi: chemical composition, biological properties and toxicity. Rev Bras Plantas Med. 2013;15(1):158-169. DOI: 10.1590/S1516-05722013000100022.
https://doi.org/10.1590/S1516-0572201300...
). S. terebinthifolia is frequently found as part of the caatinga, a vegetation biome that covers most of the Brazilian semiarid region. This plant is an important natural resource to be explored, because traditional communities living in these areas could economically and socially benefit from its production. Therefore, studies that encourage the use of S. terebinthifolia as a new source of income can help to improve the quality of life of these communities and enable their survival from natural resources in their own environment.

S. terebinthifolia has been used in folk medicine as a cicatricial and anti-inflammatory compound (Carvalho et al., 2013Carvalho M, Melo A, Aragão C, Raffin FN, Moura TFAL. Schinus terebinthifolius Raddi: chemical composition, biological properties and toxicity. Rev Bras Plantas Med. 2013;15(1):158-169. DOI: 10.1590/S1516-05722013000100022.
https://doi.org/10.1590/S1516-0572201300...
) in the treatment of oral conditions (Araujo et al., 2018Araujo GS, Santos EB, Silva PPS, Oliveira VJS, Brito NM. Ethnobotanical survey of plant species used in dentistry in the reconcavo baiano, Brazil. Senare. 2018;17(1):43-50.) and as an antimicrobial agent (Gilbert, Favoreto, 2011Gilbert B, Favoreto R. Schinus terebinthifolius Raddi. Rev Fitos. 2011;6(1):43-56.; Do Nascimento et al., 2012Do Nascimento AF, da Camara CAG, de Moraes MM, Ramos CS. Essential oil composition and acaricidal activity of Schinus terebinthifolius from Atlantic forest of Pernambuco, Brazil against Tetranychus urticae. Nat Prod Commun. 2012;7(1):129-132. DOI: 10.1177/1934578X1200700141
https://doi.org/10.1177/1934578X12007001...
). Traditional communities have used S. terebinthifolia as an extract (obtained from the inner bark, leaves, and fruits) and as an essential oil (EO) (from the leaves and fruit) (Gilbert, Favoreto, 2011Gilbert B, Favoreto R. Schinus terebinthifolius Raddi. Rev Fitos. 2011;6(1):43-56.). Recent scientific studies have investigated its properties such as antitumor and antioxidant effects (Gilbert, Favoreto, 2011Gilbert B, Favoreto R. Schinus terebinthifolius Raddi. Rev Fitos. 2011;6(1):43-56.; Bendaoud et al., 2010Bendaoud H, Romdhane M, Souchard JP, Cazaux S, Bouajila J. Chemical composition and anticancer and antioxidant activities of Schinus Molle L. and Schinus terebinthifolius Raddi berries essential oils. J Food Sci. 2010;75(6):466-72. DOI: 10.1111/j.1750-3841.2010.01711.x.
https://doi.org/10.1111/j.1750-3841.2010...
), anti-inflammatory effects (Estevão et al., 2017Estevão LRM, Simões RS, Cassini-Vieira P, Canesso MCC, Barcelos LS, Rachid MA, et al. Schinus terebinthifolius Raddi (Aroeira) leaves oil attenuates inflammatory responses in cutaneous wound healing in mice. Acta Bras Cir. 2017;32(9):726-735. DOI: 10.1590/s0102-865020170090000005
https://doi.org/10.1590/s0102-8650201700...
), and antimicrobial activity (Dannenberg et al., 2019Dannenberg GS, Funck GD, Silva WP, Fiorentini AM. Essential oil from pink pepper (Schinus terebinthifolius Raddi): Chemical composition, antibacterial activity and mechanism of action. Food Control. 2019;95:115-120. DOI:10.1016/j.foodcont.2018.07.034
https://doi.org/10.1016/j.foodcont.2018....
; Oliveira et al., 2018Oliveira MS, Gontijo SL, Teixeira MS, Texeira KIR, Takashi JA, Millan RDS, et al. Chemical composition and antifungal and anticancer activities of extracts and essential oils of Schinus terebinthifolius Raddi fruit. Rev Fitos . 2018;12(2):135-146.).

Most of these published articles used S. terebinthifolia EO obtained from ripe fruit (Bendaoud et al., 2010Bendaoud H, Romdhane M, Souchard JP, Cazaux S, Bouajila J. Chemical composition and anticancer and antioxidant activities of Schinus Molle L. and Schinus terebinthifolius Raddi berries essential oils. J Food Sci. 2010;75(6):466-72. DOI: 10.1111/j.1750-3841.2010.01711.x.
https://doi.org/10.1111/j.1750-3841.2010...
, Dannenberg et al., 2019Dannenberg GS, Funck GD, Silva WP, Fiorentini AM. Essential oil from pink pepper (Schinus terebinthifolius Raddi): Chemical composition, antibacterial activity and mechanism of action. Food Control. 2019;95:115-120. DOI:10.1016/j.foodcont.2018.07.034
https://doi.org/10.1016/j.foodcont.2018....
; Oliveira et al., 2018Oliveira MS, Gontijo SL, Teixeira MS, Texeira KIR, Takashi JA, Millan RDS, et al. Chemical composition and antifungal and anticancer activities of extracts and essential oils of Schinus terebinthifolius Raddi fruit. Rev Fitos . 2018;12(2):135-146.); thus, additional studies are required to assess whether S. terebinthifolia EO obtained from green fruit is an effective and safe source of active compounds. This was the aim of the present study, since previous investigations have already indicated differences in the chemical composition of green and ripe fruits of S. terebinthifolia (Barbosa, Demuner, Clemente, 2007Barbosa LCA, Demuner AJ, Clemente AD. Seasonal variation in the composition of volatile oils from Schinus Terebinthifolius Raddi. Quim Nova. 2007;30(8):1959-1965.; Do Nascimento et al., 2012Do Nascimento AF, da Camara CAG, de Moraes MM, Ramos CS. Essential oil composition and acaricidal activity of Schinus terebinthifolius from Atlantic forest of Pernambuco, Brazil against Tetranychus urticae. Nat Prod Commun. 2012;7(1):129-132. DOI: 10.1177/1934578X1200700141
https://doi.org/10.1177/1934578X12007001...
; Ennigrou et al., 2017Ennigrou A, Casabianca H, Laarif A, Hanchi B, Hosni K. Maturation-related changes in phytochemicals and biological activities of the Brazilian pepper tree (Schinus terebinthifolius Raddi) fruits. S Afr J Bot. 2017;108:407-415. Doi: 10.1016/j.sajb.2016.09.005
https://doi.org/10.1016/j.sajb.2016.09.0...
). Moreover, it is known that different proportions of chemical constituents might give rise to different pharmacological effects. No study has analyzed the EO obtained from S. terebinthifolia green fruit collected from the caatinga biome of Brazil. Therefore, the present study aimed to assess the chemical composition of the EO of S. terebinthifolia green fruit and to evaluate its antimicrobial activity, cytotoxicity, and systemic toxicity.

MATERIAL AND METHODS

Plant material

Green fruits of S. terebinthifolia were collected in the semi-arid region of Campina Grande, Paraíba, Brazil (7º12 ‘35 “S, 35º 54’ 57” W). The specimens were deposited in the collection of the Herbarium Manuel de Arruda Câmara (ACAM), State University of Paraiba (UEPB), Campus I, Campina Grande, Paraíba, under registry number 486/ACAM.

Essential oil

Hydrodistillation was performed in a Clevenger type system, at a proportion of 1:4 (m/v), with a distillation time of 2 h.

Essential oil fractions

Chemical compounds were initially monitored by thin layer chromatography (TLC) with silica gel matrix (TLC silica gel 60 F254 - Merck® - Darmstadt, Germany). A mixture of ethyl acetate (Synth® - São Paulo, Brazil) and hexanoic acid (Synth® - São Paulo, Brazil) (85:15 v/v) was used as mobile phase. The components were revealed using anisaldehyde chromate. Fractionation of the EO was carried out on solid-liquid silica phase (Merck® - Darmstadt, Germany) with a porous plate funnel-filter chromatographic column according to the gradient of polarity: hexane (1x100mL), hexane:ethyl acetate ratio of 98:2, 96:4, 96:4/92:8, 90:10, 88:12, 86:14, 84:16, 82:18, and 80:20 (v/v). Fractions were monitored by TLC and pooled according to their similarity profile and then concentrated under vacuum (Rotavapor R-215, BUCHI® - São Paulo, Brazil). The result was six final fractions that were tested for their antiproliferative activity against human tumor cell lines

Phytochemical characterization

The EO was chemically identified using a gas chromatograph (GC) (QP 2010 Plus, Shimadzu Co, São Paulo, Brazil) coupled to a mass spectrometer (MS) equipped with a DB-5 capillary column (30m x 0.25μm x 2.5m) (J&W Scientific - California, USA) and a scanning mode detector (40 - 400 m/z). The EO (400 μL) was inserted into glass vials, and 1.0 mL of trimethylsilyl for silanization solution were added. The temperature program was 60°C (0.3 min), followed by 240°C (15 min), at an increase of 3°C/min. The sample (0.5 μL) was injected by an auto-injector on the split-less mode, and integration was performed using a specific software from the equipment. The analytes were identified by comparison using the database of the equipment (NIST library), and the literature (Adams, 2007Adams RP. Identification of essential oil components by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation: Carol Stream, 2007.). Additionally, it was conducted a comparison with the data obtained from GC-MS (retention index and fragmentation time) of authentic patterns under the same conditions.

Evaluation of antimicrobial activity

The minimum inhibitory concentration (MIC), bactericidal concentration (MBC) and fungicidal concentration (MFC) (CLSI, 2008CLSI. Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts - M27-A3, Wayne, PA, USA , 2008;28(14):13.; CLSI, 2009CLSI. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. M07-A8, Wayne, PA, USA, 2009;29(1).) were obtained for the following microorganisms: Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11775), Salmonella enteritidis (ATCC 13076), Candida parapsilosis ATCC 22019, C. tropicalis ATCC 750, C. glabrata ATCC 90030, C. krusei ATCC 34135, C. albicans (ATCC 10231), C. albicans (ATCC 5314), and two clinical strains of C. albicans obtained from oral candidiasis lesions from volunteers. All ethical aspects were respected (process number: 51779315.7.0000.5187).

The microdilution method was applied. Bacteria were grown in Müller-Hinton broth (Kasvi® Paraná, Brazil) and yeast were grown in RPMI 1640 (Sigma-Aldrich® - Missouri, EUA). The EO serially was diluted (2000 to 0.4882μg/mL) in the plates wells. Subsequently, bacterial (5 × 105 colony forming units - (CFU)/mL) and fungal (2.5 × 103 CFU/mL) suspensions were added and the plates were incubated at 37°C for 24 h. Chloramphenicol and nystatin (both 500μg/mL) (Sigma-Aldrich®) were used as pharmacological controls. Visible microbial growth was confirmed with triphenyl tetrazolium chloride for bacteria and by the changing coloration of the RPMI 1640 medium for yeast. After 24 h, 50μL of each well, with equal and/or higher MICs were sub-cultured (37ºC, 24h) on brain heart infusion agar (Sigma-Aldrich®) (bacteria) or sabouraud dextrose agar (Kasvi®) (yeast).

Inhibition of Candida albicans biofilm

The biofilm assay was performed with the previously stated C. albicans strains. C. albicans suspensions (1×107CFU/mL) were prepared in RPMI 1640 (Sigma-Aldrich®) and 100μL was distributed to each well of a 96-well microplate and incubated at 37°C for 24 h. The biofilms were then exposed to EO at concentrations based on the MIC (1000 μg MIC, 2000 μg 2×MIC, and 4000 μg 4×MIC). Nystatin was used as a control. After treatment, the biofilms were seeded onto saboraud dextrose agar plates (Kasvi®) to assess the number of viable microorganisms (Silva et al, 2019bSilva DR, Rosalen PL, Freires IA, Sardi JCO, Lima RF, Lazarini JG, et al. Anadenanthera Colubrina vell Brenan: anti-Candida and antibiofilm activities, toxicity and therapeutical action. Braz Oral Res. 2019b;33,e023. DOI: 10.1590/1807-3107bor-2019.vol33.0023
https://doi.org/10.1590/1807-3107bor-201...
).

In vitro antiproliferative activity evaluation

The antiproliferative activity was evaluated against a panel of eight human tumor cell lines [U251 (glioblastoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multi-drug resistant ovarian adenocarcinoma), 786-0 (kidney, adenocarcinoma), NCI-H460 (lung, large cell carcinoma), PC3 (prostate, adenocarcinoma), HT-29 (colon, adenocarcinoma), and K562 (chronic myeloid leukemia)] and one non-tumor human cell line (HaCaT, immortalized keratinocytes). The tumor cell lines were kindly provided by the Frederick Cancer Research & Development Center, National Cancer Institute, Frederick, MA, USA. The non-tumor cell line was provided by Dr. Ricardo Della Coletta (University of Campinas). Stock cultures were grown in 5 mL of complete medium [RPMI-1640 (Gibco, USA)] supplemented with 5% fetal bovine serum (FBS, Gibco, USA, catalog number 16000044) and 1% penicillin-streptomycin mixture [1000 U∙mL- 1:1000 µg·mL-1 (Vitrocell, Brazil)] at 37ºC in a 5% CO2 humidified atmosphere. All experiments were conducted with cells grown for 5 to 12 passages.

The EO and its fractions were first diluted to 100 mg/mL in DMSO (Merck - Darmstadt, Germany), followed by serial dilution in complete RPMI 1640 medium, affording the final EO concentrations of 0.25, 2.5, 25, and 250 μg/mL. Twenty-four hours after being transferred to 96-well plates (inoculation density: 3 to 6 x 104 cell/mL, 100 μL/well), all cell lines were exposed to freshly diluted EO samples, in triplicate (100 μL/well), for 48 h at 37 °C under 5% CO2. Doxorubicin (0.025, 0.25, 2.5, and 25 μg/mL, 100 μL/well) was used as a positive control. Before the 48 h exposure, one control plate (T0) containing cells for each tested cell line was utilized for cell fixation using trichloroacetic acid (TCA, 50%, 50 μL/well) (Sigma-Aldrich) to establish the initial cell amount. After the 48 h exposure, treated cells were fixed with 50% TCA for 1 h at 4°C and then washed and kept at room temperature until completely dry. The cellular protein content was then stained with sulforhodamine B dye (0.4% in 1% acetic acid, 50 μL/well) (Sigma-Aldrich), and the bound dye was solubilized with Trizma base solution (10 μM, pH 10.5, 150 μL/well) (Sigma-Aldrich). The spectrophotometric reading was performed at 540 nm with a VersaMax plate reader (Molecular Devices).

Based on the cell absorbance before (T0) and after (T1) EO exposure, the cell growth (%) for each tested EO sample against each cell line was calculated by the following equations: 100×[(T-T0)/(T1-T0)] when T > T0; and 100×[(T-T0)/T0)] when T ≤ T0. T is the mean absorbance of treated cells, T1 is the mean absorbance of untreated cells after 48 h of exposure, and T0 is the mean absorbance of untreated cells at the beginning of the 48 h exposure (Monks et al., 1991Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diversepanel of cultured human tumor cell lines. J Natl Cancer Instit. 1991;83(11):757-766. DOI: 10.1093/jnci/83.11.757
https://doi.org/10.1093/jnci/83.11.757...
; Shoemaker, 2006Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer. 2006;6(10):813-823.). Based on these results, the sample concentration required to achieve total growth inhibition (TGI) was calculated by sigmoidal regression using Origin 8.0 software.

The selectivity index (SI) was calculated according to Eq. (1).

S I H a C a t c a n c e r l i n e c o m p u n d = G I 50 G I 50 c a n c e r l i n e (1)

Cytotoxicity against RAW 264.7 macrophage

A suspension of RAW 264.7 macrophages (1x105 cells/mL) (ATTC, Manassa, VA, USA) was prepared in RPMI 1640 (Sigma-Aldrich®) containing 5% FBS (Gibco® BRL - Dublin, Ireland) and 2 mg/mL penicillin-streptomycin (Sigma-Aldrich®). Aliquots of 100 µl of suspension were applied to each well of the 96-well plates and incubated for 24 h at 37° C under 5% CO2, a control plate was also prepared (T0 plate). The EO was initially diluted in DMSO, followed by serial dilution on RPMI 1640 (250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0.24 μg/mL) (T-plate). The plates were incubated for 48 h at 37°C in 5% CO2.

Previous to EO sample addition, the cells in the T0 plate were fixed with 50% TCA (50 μL/well) to determine the cell quantity at the time of EO addition. After 48 h, the T-plate cells were fixed with 50% TCA and incubated for 1 hour, followed by washing with distilled water to remove any residue. After the plates were dried at room temperature, 50 μL of 0.4% sulforhodamine B protein (SRB) diluted in 1% acetic acid as added to the wells. The plates were held at room temperature for 30 minutes and then washed 4 times with 1% acetic acid to remove the dye that had not attached to the cells. Dye that had bound to the cellular proteins was solubilized with 150 μL/well Trizma base solution (10 μM, pH 10.5). The absorbance was read at 540 nm (VersaMax, Molecular Devices), and viability was calculated by the following equation: cell viability (%) = (sample/control) x 100, where sample and control are equivalent to the absorbance of the wells with and without the addition of treatment, respectively.

In vitro toxicity against human erythrocytes

The hemolysis assay was performed as previously described (Luize et al., 2005Luize PS, Tiuman TS, Morello LG, Maza PK, Ueda-Nakamura T, Filho BPD, et al. Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi. Braz J Pharm Sci. 2005;41(1):85-94. DOI: 10.1590/S1516-93322005000100010
https://doi.org/10.1590/S1516-9332200500...
). A 5% suspension of erythrocytes was mixed with different concentrations of the EO (16, 8, 4, 2 and 1 mg/mL). After incubating for 1 h at room temperature, the samples were centrifuged (500 g, 10 min), and the absorbance at 540 nm was read by a spectrophotometer (UV mini - 1240 - Shimadzu® - São Paulo, Brazil). The level of hemolysis was scored as follows: (-) 0% hemolysis; (+) 1 - 25% hemolysis; (++) 26 - 50% hemolysis; (+++) 51 - 75% hemolysis; and (++++) 76 - 100% hemolysis. A saline-treated erythrocyte suspension was used as a negative control. Turk’s liquid (2% acetic acid) plus methylene blue was used as a positive control.

Systemic toxicity test in Galleria mellonella model

The EO was evaluated in vivo to assess its acute systemic toxicity using the Galleria mellonella larvae model (Rochelle et al., 2016Rochelle SLA, Sardi JCO, Freires IA, Galvão LCC, Lazarini JG, Alencar SM, et al. The anti-biofilm potential of commonly discarded agro-industrial residues against opportunistic pathogens. Ind Crop Prod. 2016;87:150-160. DOI: 10.1016/j.indcrop.2016.03.044
https://doi.org/10.1016/j.indcrop.2016.0...
). Doses of the EO (50 to 10g/kg) in were used to determine the LD50. Ten larvae were randomly selected for each group. 5 μL of each EO concentration diluted in 20% DMSO (Merck® - Darmstadt, Germany) was added to test and control groups. The larvae were incubated at 30°C, and their survival was evaluated every 9 h up to 48 h. The larvae that did not move and that presented melanization were considered as dead.

Statistical analysis

Analyses were performed in triplicate and in three independent experiments. The data were initially evaluated to determine their distribution using the Kolmogorov - Smirnov test. One way analysis of variance (ANOVA) and the post-hoc Tukey test were applied at a significance level of 5% (α<0.05).

RESULTS AND DISCUSSION

In accordance with Holetz et al. (2002Holetz FB, Pessini GL, Sanches NR, Cortez DA, Nakamura CV, Filho BP. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz. 2002;97(7):1027-1031. DOI: 10.1590/ S0074-02762002000700017
https://doi.org/10.1590/ S0074-027620020...
), the EO presented weak activity against all strains of C. albicans (MIC 1000 μg/mL), and no evidence was observed of growth inhibition for the non-albicans strains, or for the bacterial species tested (MIC > 1000 μg/mL) (Table I). Moura et al. (2011Moura TFAL, Raffin FN, Santos ALR. Evaluation of a preservative system in a gel containing hydroalcoholic extract of Schinus terebinthifolius. Rev Bras Farmacogn. 2011;21(3):532-6. DOI: 10.1590/S0102-695X2011005000102
https://doi.org/10.1590/S0102-695X201100...
) found limited antimicrobial activity for S. terebinthifolia, similar to the findings of the present study. In the Moura study, the authors analyzed a gel containing S. terebinthifolia extract against Candida and bacteria species, and no significant decrease in the microbial load was observed.

TABLE I
Minimum Inhibitory Concentration (MIC), Minimum Fungicidal (MFC) and Minimum Bactericidal (MBC) concentrations of the S. terebinthifolia green fruits EO, according to different microbial species

In contrast to the findings of the current study, favorable antimicrobial results were reported for the EO obtained from S. terebinthifolia ripe fruits against fungus (Oliveira et al., 2018Oliveira MS, Gontijo SL, Teixeira MS, Texeira KIR, Takashi JA, Millan RDS, et al. Chemical composition and antifungal and anticancer activities of extracts and essential oils of Schinus terebinthifolius Raddi fruit. Rev Fitos . 2018;12(2):135-146.) and gram-positive and gram-negative bacteria (Dannenberg et al., 2019Dannenberg GS, Funck GD, Silva WP, Fiorentini AM. Essential oil from pink pepper (Schinus terebinthifolius Raddi): Chemical composition, antibacterial activity and mechanism of action. Food Control. 2019;95:115-120. DOI:10.1016/j.foodcont.2018.07.034
https://doi.org/10.1016/j.foodcont.2018....
). The differing results can be attributed to differences in the chemical composition between ripe and green fruits, which was demonstrated by Ennigrou et al. (2017Ennigrou A, Casabianca H, Laarif A, Hanchi B, Hosni K. Maturation-related changes in phytochemicals and biological activities of the Brazilian pepper tree (Schinus terebinthifolius Raddi) fruits. S Afr J Bot. 2017;108:407-415. Doi: 10.1016/j.sajb.2016.09.005
https://doi.org/10.1016/j.sajb.2016.09.0...
). The authors concluded that the plant maturation process resulted in a significant change in its chemical composition. They stated that these changes were reflected in the antimicrobial activity such that gram-positive and gram-negative bacteria were more susceptible to the ripe fruit EO than to the green fruit EO.

C. albicans is a susceptible yeast when in a planktonic form. However, in the biofilm assay, C. albicans MYA 2876 and the two clinical strains showed no reduced viability (expressed in CFU) upon EO exposure. A small but significant reduction was seen in the viability of C. albicans ATCC 10231 (Figure 1). These results may be associated with the limited penetration of the S. terebinthifolia EO into the biofilm structure. Biofilm formation is considered one of the main virulence factors associated with C. albicans. Additionally, cells within the biofilms are protected from environmental stresses including host immune defenses and antifungal treatment, which carries important clinical consequences for the treatment of biofilm-associated infections (Wall et al., 2019Wall G, Montelongo-Jauregui D, Vidal Bonifacio B, Lopez-Ribot JL, Uppuluri P. Candida albicans biofilm growth and dispersal: contributions to pathogenesis. Curr Opin Microbiol . 2019;52:1-6. DOI:10.1016/j.mib.2019.04.001.
https://doi.org/10.1016/j.mib.2019.04.00...
).

FIGURE 1
Anti-biofilm activity of S. terebinthifolia green fruits EO at concentrations 1000 μg/mL (MIC), 2000 μg/mL (twice MIC) and 4000 μg/mL (four times MIC) on C. albicans strains.

The EO extracted from S. terebinthifolia green fruits had a yield of 1 mL. Its chemical composition is presented in Table II. The major constituints of S. terebinthifolia green fruits EO are diterpenes: alpha-feladrene (37.05%), beta-feladrene (24.10%), alpha-pinene (15.91%) and germacrene D (14.47%). Do Nascimento et al. (2012Do Nascimento AF, da Camara CAG, de Moraes MM, Ramos CS. Essential oil composition and acaricidal activity of Schinus terebinthifolius from Atlantic forest of Pernambuco, Brazil against Tetranychus urticae. Nat Prod Commun. 2012;7(1):129-132. DOI: 10.1177/1934578X1200700141
https://doi.org/10.1177/1934578X12007001...
) analyzed the chemical composition of S. terebinthifolia green fruit EO, and alpha-phellandrene was one of the major compounds identified, but in a different proportion from that identified in the current study. Furthermore, unlike the present study, other compounds were previously identified, such as limonene and beta-pinene. These differences might be associated with changes in the conditions to which the plants were subjected; Do Nascimento et al. (2012)Do Nascimento AF, da Camara CAG, de Moraes MM, Ramos CS. Essential oil composition and acaricidal activity of Schinus terebinthifolius from Atlantic forest of Pernambuco, Brazil against Tetranychus urticae. Nat Prod Commun. 2012;7(1):129-132. DOI: 10.1177/1934578X1200700141
https://doi.org/10.1177/1934578X12007001...
collected plants from a region of Atlantic rainforest in the northeast of Brazil, which is a different biome from caatinga. Previous studies have also indicated a seasonality effect in the chemical compositions of natural compounds (Macedo et al., 2020Macedo DG, Souza MMA, Morais-Braga MFB, Coutinho HDM, Santos ATL, Machado AJT, et al. Seasonality influence on the chemical composition and antifungal activity of Psidium myrtoides O. Berg. S Afr J Bot . 2020;128:9-17. DOI: 10.1016/j.sajb.2019.10.009
https://doi.org/10.1016/j.sajb.2019.10.0...
; Bitu et al., 2015Bitu VCN, Costa JGM, Rodrigues FFG, Colares AV, Coutinho HDM, Botelho MA, et al. Effect of Collection Time on Composition of Essential Oil of Schauer (Verbenaceae) Growing in Northeast Brazil. J Essent Oil-Bear Plants. 2015;18(3):647-653.).

TABLE II
Analyses identified in the essential oil of S. terebinthifolia green fruits EO, by gas chromatography with mass spectrometry

Ennigrou et al. (2017Ennigrou A, Casabianca H, Laarif A, Hanchi B, Hosni K. Maturation-related changes in phytochemicals and biological activities of the Brazilian pepper tree (Schinus terebinthifolius Raddi) fruits. S Afr J Bot. 2017;108:407-415. Doi: 10.1016/j.sajb.2016.09.005
https://doi.org/10.1016/j.sajb.2016.09.0...
) compared the chemical compositions of S. terebinthifolia EO obtained from green and ripe fruits. The authors found alpha-phellandrene, alpha-pinene, and germacrene D among the major compounds in the green fruit EO, similar to the present findings. Additionally, the authors concluded that a significant change was seen in the chemical profile of the S. terebinthifolia fruit after the maturation process. Para-cymene and germacrene D were more abundant in the EO obtained from green fruit, while a sharp decrease in alpha-pinene and beta-phellandrene was noted in the ripe fruit EO. The differing chemical compositions of ripe and green fruit EO might explain the different biological activities of the EOs. Indeed, the present study was performed to investigate this issue, because most of the previously published articles used EO extracted from ripe fruit.

The antiproliferative activity (Table III, Figure 2) was assessed using the protocol developed by the Frederick Cancer Research & Development Center, National Cancer Institute (Shoemaker, 2006Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer. 2006;6(10):813-823.) using doxorubicin (Table III, Figure 2A) as a positive control. According to Fouche et al. (2008Fouche G, Cragg GM, Pillay P, Kolesnikova N, Maharaj VJ, Senabe J. In vitro anticancer screening of South African plants. J Ethnopharmacol. 2008;119(3):455-461. DOI: 10.1016/j.jep.2008.07.005
https://doi.org/10.1016/j.jep.2008.07.00...
), the antiproliferative activity of compounds can be classified as inactive (TGI ≥ 50 μg/mL), weak (15 μg/mL ≤ TGI < 50 μg/mL), moderate (6.25 μg/mL ≤ TGI < 15 μg/mL), or potent (TGI < 6.25 μg/mL). Based on the concept of the therapeutic index described by Muller and Milton (2012Muller PY, Milton MN. The determination and interpretation of the therapeutic index in drug development. Nat Rev Drug Discov. 2012;11(10):751-761. DOI:10.1038/nrd3801
https://doi.org/10.1038/nrd3801...
), we calculated the selective index (SI) by determining the ratio between the TGI value for a non-tumor cell line (HaCaT, immortalized human keratinocytes) and the TGI value for each tumor cell line. The SI indicates the potential antiproliferative effect against normal tissues such as mucosa and bone marrow (i.e., an on-target toxicology approach).

TABLE III
Antiproliferative activity S. terebinthifolia green fruits EO and its fractions against human tumor and non-tumor cell lines expressed as concentration required to elicit total growth inhibition (TGI, μg/ml)

Using these criteria, the S. terebinthifolia EO (Table III, Figure 2B) and the hexane:ethyl acetate fraction 96:4 (Table III, Figure 2C) were considered as inactive, showing nonspecific cell death up to 250 μg/mL. The most active fraction, the hexane:ethyl acetate fraction 80:20, exhibited potent activity (TGI < 0.25 μg/mL, Table III, Figure 2D) against almost all tumor cell lines and against the non-tumor cell line (HaCaT).

The hexane:ethyl acetate fraction 90:10 (Table III, Figure 2E) showed potent and selective cytostatic effects against leukemia (K562, TGI = 1.3 μg/mL, SI = 7.2), glioblastoma (U251, TGI = 1.4 μg/mL, SI = 6.5), kidney (786-0, TGI = 2.0 μg/mL, SI = 4.6), and prostate cell lines (PC-3, TGI = 4.2 μg/mL, SI = 2.2). The hexane:ethyl acetate fraction 86:14 (Table III, Figure 2F) was as potent as the hexane:ethyl acetate fraction 90:10 against the leukemia (K562, TGI = 1.4 μg/mL, SI = 9.4) cell line despite being less active than the hexane:ethyl acetate fraction 90:10 against U251, 786-0, and PC-3 cells. Both the hexane:ethyl acetate fractions 90:10 and 86:14 showed moderate to weak cytostatic effects against the other tumor cell lines (Table III).

Further, the hexane:ethyl acetate fraction 94:6/92:8 (Table III, Figure 2G) showed weak and low selective cytostatic effects against glioblastoma (U251, TGI = 42.7 μg/mL, SI = 1.5), prostate (PC-3, TGI = 43.2 μg/mL, SI = 1.5), and leukemia tumor cell lines (K562, TGI = 48.1 μg/mL, SI = 1.3). Finally, the hexane:ethyl acetate 98:2 fraction (Table III, Figure 2H) showed cytostatic effects ranging from potent (K562 and 786-0) to moderate (U251) and weak (MCF-7, NCI-ADR/RES, and HT29), while it was inactive against the HCI-H460 and PC-3 cell lines.

Previous studies have demonstrated the antiproliferative effect of S. terebinthifolia EO obtained from ripe fruits (Bendaoud et al., 2010Bendaoud H, Romdhane M, Souchard JP, Cazaux S, Bouajila J. Chemical composition and anticancer and antioxidant activities of Schinus Molle L. and Schinus terebinthifolius Raddi berries essential oils. J Food Sci. 2010;75(6):466-72. DOI: 10.1111/j.1750-3841.2010.01711.x.
https://doi.org/10.1111/j.1750-3841.2010...
; Silva et al., 2017Silva BG, Fileti AMF, Foglio MA, Ruiz ALTG, Rosa PTV. Supercritical carbono dioxide extraction of compounds from Schinus terebinthifolius Raddi fruits: Effects of operating conditions on global yield, volatile compounds, and antiproliferative activity against human tumor cell lines. J Supercrit Fluid. 2017;130:10-16. DOI: 10.1016/j.supflu.2017.07.006
https://doi.org/10.1016/j.supflu.2017.07...
; Silva et al., 2019aSilva BG, Foglio MA, Rosa PTV, Taranto OP, Fileti AMF. Optimization of hydrodistillation and In vitro anticancer activity of essential oil from Schinus terebinthifolius Raddi fruits. Chem Eng Com. 2019a;206(5):619-629, DOI: 10.1080/00986445.2018.1515074
https://doi.org/10.1080/00986445.2018.15...
).

Silva et al. (2017Silva BG, Fileti AMF, Foglio MA, Ruiz ALTG, Rosa PTV. Supercritical carbono dioxide extraction of compounds from Schinus terebinthifolius Raddi fruits: Effects of operating conditions on global yield, volatile compounds, and antiproliferative activity against human tumor cell lines. J Supercrit Fluid. 2017;130:10-16. DOI: 10.1016/j.supflu.2017.07.006
https://doi.org/10.1016/j.supflu.2017.07...
) and Silva et al. (2019a)Silva BG, Foglio MA, Rosa PTV, Taranto OP, Fileti AMF. Optimization of hydrodistillation and In vitro anticancer activity of essential oil from Schinus terebinthifolius Raddi fruits. Chem Eng Com. 2019a;206(5):619-629, DOI: 10.1080/00986445.2018.1515074
https://doi.org/10.1080/00986445.2018.15...
used different methods of extraction (supercritical CO2 extraction and optimized hydrodistillation using a Clevenger apparatus, respectively), and they identified similar compounds as in the current study of green fruit EO, such as alpha-phellandrene, alpha-pinene, and germacrene D, but in different proportions. The EO in Silva et al. (2019a)Silva BG, Foglio MA, Rosa PTV, Taranto OP, Fileti AMF. Optimization of hydrodistillation and In vitro anticancer activity of essential oil from Schinus terebinthifolius Raddi fruits. Chem Eng Com. 2019a;206(5):619-629, DOI: 10.1080/00986445.2018.1515074
https://doi.org/10.1080/00986445.2018.15...
showed potent activity for all cell lines investigated, with promising results against leukemia, kidney, multidrug-resistant ovarian, and prostate tumor cell lines. Silva et al. (2017)Silva BG, Fileti AMF, Foglio MA, Ruiz ALTG, Rosa PTV. Supercritical carbono dioxide extraction of compounds from Schinus terebinthifolius Raddi fruits: Effects of operating conditions on global yield, volatile compounds, and antiproliferative activity against human tumor cell lines. J Supercrit Fluid. 2017;130:10-16. DOI: 10.1016/j.supflu.2017.07.006
https://doi.org/10.1016/j.supflu.2017.07...
found potent activity against multidrug-resistant ovarian, prostate, and ovarian tumor cell lines. Both studies discussed the possible association between the chemical compounds identified in the EO and the antiproliferative activity.

Bendaoud et al. (2010Bendaoud H, Romdhane M, Souchard JP, Cazaux S, Bouajila J. Chemical composition and anticancer and antioxidant activities of Schinus Molle L. and Schinus terebinthifolius Raddi berries essential oils. J Food Sci. 2010;75(6):466-72. DOI: 10.1111/j.1750-3841.2010.01711.x.
https://doi.org/10.1111/j.1750-3841.2010...
) demonstrated an antiproliferative effect of the EO against human breast cancer cells (MCF-7). The authors found alpha-phellandrene, alpha-pinene, and germacrene D as some of the major compounds in the EO, similar to this study. The authors attributed the antiproliferative activity of the EO to the presence of sesquiterpenes, such as germacrene D. Additionally, other compounds present in the unripe fruit EO analyzed in the present study have been associated with antiproliferative effects. For example, alpha-phellandrene has been described as inducing tumor cell necrosis through ATP depletion in human tumor liver cells (Hsieh et al., 2014Hsieh SL, Li YC, Chang WC, Chung JG, Hsieh LC, Wu CC. Induction of necrosis in human liver tumor cells by α-phellandrene. Nutr Cancer. 2014;66(6):970-979. DOI:10.1080/01635581.2014.936946
https://doi.org/10.1080/01635581.2014.93...
), and alpha-pinene (the third-most abundant compound identified in the present study) has been described as an apoptosis inductor in malignant melanoma cells (Matsuo et al., 2011Matsuo AL, Figueiredo CR, Arruda DC, Pereira FV, Scutti JAB, Massaoka MH, et al. α-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model. Biochem Bioph Res Co. 2011;411(29);449-454. DOI: 10.1016/j.bbrc.2011.06.176
https://doi.org/10.1016/j.bbrc.2011.06.1...
).

FIGURE 2
Cell lines proliferation according to the concentration of positive control Doxorubicin (Figure 2A), S. terebinthifolia green fruits EO (Figure 2B) and its hexane: ethyl acetate fractions (Figure 2C-2H).

The hemolysis test, which showed 50% hemolysis at 16 mg/mL. (Table IV), and the antiproliferative activity against keratinocytes represent initial safety data for the use of this EO. Erythrocyte membrane stability is assessed in toxicity screening and is an indicator of in vitro damage, especially when associated with oxidative stress (Pathak, Sharma, Shrivastva, 2012Pathak AK, Sharma S, Shrivastva P. Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate. J Appl Oral Sci. 2012;20(1):70-5. DOI: 10.1590/S1678-77572012000100013.
https://doi.org/10.1590/S1678-7757201200...
). The results from the Galleria mellonella larvae model suggest a good safety profile, since only high doses (40g/kg) affected the larvae viability, resulting in a 10% death rate (Figure 3). This methodology has been gaining in prominence due to its applicability in toxicological testing and the efficacy for applying it to new compounds (Freires et al., 2017Freires IA, Sardi JCO, de Castro RD, Rosalen PL. Alternative Animal and Non-Animal Models for Drug Discovery and Development: Bonus or Burden? Pharm Res. 2017;34(4):681-686. DOI: 10.1007/s11095-016-2069-z
https://doi.org/10.1007/s11095-016-2069-...
).

TABLE IV
Percentage distribution of the hemolysis produced by the S. terebinthifolia green fruits EO, according to the concentrations

FIGURE 3
In vivo toxicity of S. terebinthifolia green fruits EO in invertebrate Galleria mellonella model.

According to the International Organization for Standardization (ISO) publication 10993-5 (ISO, 2009International Standard Organization (ISO - 10993-5). Biological evaluation of medical devices -Part 5: Tests for in vitro cytotoxicity. 2009;3:1-42), concentrations that reduce cell viability to less than 70% are considered cytotoxic. S. terebinthifolia green fruits EO resulted in high cell viability grater than 85% for RAW 264.7 macrophages exposed to concentrations of 62.5μg/mL (Figure 4). These macrophage viability results could guide future studies to evaluate the anti-inflammatory potential of this EO.

FIGURE 4
Cell viability percentage of RAW 264.7 macrophages lineage according to the concentration of S. terebinthifolia green fruits EO.

Although S. terebinthifolia is as an antimicrobial agent in folk medicine (Do Nascimento et al., 2012Do Nascimento AF, da Camara CAG, de Moraes MM, Ramos CS. Essential oil composition and acaricidal activity of Schinus terebinthifolius from Atlantic forest of Pernambuco, Brazil against Tetranychus urticae. Nat Prod Commun. 2012;7(1):129-132. DOI: 10.1177/1934578X1200700141
https://doi.org/10.1177/1934578X12007001...
), the results obtained in the present study showed low antimicrobial and antibiofilm activity. Therefore, additional studies should be conducted to further investigate its use as an effective compound against microorganisms of clinical relevance. However, it presented low cytotoxic potential as demonstrated in the in vitro and in vivo models. Considering these results and the phytochemical characteristics of this EO, other properties could be considered, such as anti-inflammatory activity (Estevão et al., 2017Estevão LRM, Simões RS, Cassini-Vieira P, Canesso MCC, Barcelos LS, Rachid MA, et al. Schinus terebinthifolius Raddi (Aroeira) leaves oil attenuates inflammatory responses in cutaneous wound healing in mice. Acta Bras Cir. 2017;32(9):726-735. DOI: 10.1590/s0102-865020170090000005
https://doi.org/10.1590/s0102-8650201700...
). Furthermore, additional in vitro and in vivo assays are needed to assess pharmacological efficacy.

CONCLUSION

The EO from S. terebinthifolia green fruits showed low antimicrobial and antibiofilm activity. The major compounds identified were diterpenes, including alpha-phellandrene, beta-phellandrene, alpha-pinene, and germacrene D. Some EO fractions showed moderate antiproliferation activity against tumor cell lines, and no evidence of cytotoxic activity was identified.

ACKNOWLEDGEMENTS

The authors thank Adilson Sartoratto (lab technician), from Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA), University of Campinas (UNICAMP), for the assistance in S. terebinthifolia essential oil chemical analysis. This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brazil (CNPq), and Fundação de Apoio à Pesquisa do Estado da Paraíba - Brazil (FAPESQ).

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Publication Dates

  • Publication in this collection
    02 Dec 2022
  • Date of issue
    2022

History

  • Received
    04 May 2020
  • Accepted
    07 Dec 2020
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br