Caracterização química, atividade antimicrobiana e toxicidade dos óleos essenciais da Pimenta dioica L. e Citrus sinensis L. Osbeck Chemical characterization, antimicrobial activity and toxicity of essential oils of Pimenta dioica L. and Citrus sinensis L. Osbeck Caracterización química, actividad antimicrobiana y toxicidad de los aceites esenciales de Pimenta dioica L. y Citrus sinensis L. Osbeck Recebido: 24/05/2020 | Revisado: 25/05/2020 | Aceito: 29/05/2020 | Publicado: 15/06/2020

This study evaluated the toxicity and antimicrobial activity in the face of Escherichia coli and Staphylococcus aureus of essential oils of Pimenta dioica Lindl. and Citrus sinensis L. The essential oils (EOs) were extracted by hydrodistillation, with chemical characterization by gas chromatography coupled and mass spectrometry (GC-MS). Physicochemical parameters were determined according to the Brazilian Pharmacopeia. The toxicity test followed the bioassay with Artemia salina Leach, the EOs approved in this assay followed to evaluate its biological properties. The antimicrobial activity followed the methodology described by the Clinical and Laboratory Standards Institute using the Disc Diffusion Method, Broth Dilution for Minimum Inhibitory Concentration (MIC) and subsequent minimum bactericide concentration for to evaluate the action of EOs against E. coli and S. aureus. Both EOs showed low toxicity, and thus were evaluated for the biological antimicrobial properties. Both EOs presented bactericidal potential against the microorganisms tested, showing satisfactory results for their action. The results indicate that the evaluated EOs are composed of substances that provide and encourage their application due to their potential for antimicrobial biological activity. Research, Society and Development, v. 9, n. 7, e803974842, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4842 4

is given greater prominence as a spice, but it is also widely used for the treatment of certain diseases because it has antihypertensive, anti-inflammatory, analgesic, antimicrobial and antioxidant properties (Paula et al., 2010). The EO of C. sinensis can be classified as a mixture of terpenes, hydrocarbons and oxygenated compounds, considered chemically unstable. Sweet orange EO consists of approximately 98% R-limonene and the remaining 2% refers to a mixture of other terpenes and alpheratic aldehydes (Galvão et al. 2015).
Over the last few years natural alternatives to synthetic products have been sought, natural products are an option with less toxicity compared to other synthetic products. Thus, the present study chemically characterized, evaluated the toxicity and antimicrobial activity of The EOs of P. dioica and C. sinensis, with the perspective of offering a natural alternative to the use of synthetic antimicrobials.

Plant material
The sheets of P. dioica L. used in this study are recorded in the botanical archives of the Biodynamic Institute (IBD) of Botucatu according to a certificate in CA021205. The barks of C. sinensis L were recorded at the Federal Institute of Maranhão by the fruit and vegetable sector, such as D-25 (sweet orange, variation: pear).

Obtaining essential oils
For the extraction of the EO, the hydrodistillation technique was used with a glass Clevenger extractor coupled to a round bottom balloon packed in an electric blanket as a heat generating source. We used 30g of the dried leaves of P. dioica and 120g of the barks C. sinensis, adding distilled water (1:10). Hydrodistillation was conducted at 100°C for 5h and the extracted EO was collected. Each EO was dried by percolation with anhydrous sodium sulfate (Na2SO4) and centrifuged. These operations were performed in triplicates and samples stored in amber glass ampoules under 4°C refrigeration. Subsequently submitted the analyses.
The physicochemical parameters of the EOs were determined: density, solubility, color and appearance according to the Farmacopeia Brasileira (Farmacopeia Brasileira, 2019).

Análises Químicas
The constituents of The EOs were identified by gas chromatography coupled to mass spectrometry (CG-MS) in the Analytical Center of the Institute of Chemistry of the State University of Campinas.
1.0 mg of the sample was dissolved in 1000 μL of dichloromethane (purity 99.9%).
The conditions of analysis were as follows: Method: Adams. M, m; Injected volume: 0.3 μL; Column : Capillary HP-5MS (5% diphenyl, 95% dimethyl polysiloxane ) (Equivalent DB- System) program was used to identify the compounds in the sample.

Toxicity
For the evaluation of the lethality of Artemia salina Leach, a stock saline solution of each EO was prepared at the concentration of 10,000 mg L -1 and 0.02 mg of Tween 80 (active tense). Aliquots of 5, 50 and 500 μL of this were transferred to test tubes and completed with saline solution previously prepared up to 5 mL, obtaining concentrations of 10, 100 and 1000 mg L -1 , respectively. All tests were performed in triplicates, where ten larvae in the nauplium phase were transferred to each of the test tubes.
For white control, 5 mL of saline solution was used for positive control K2Cr2O7 and for negative control 5 mL of a solution 4 mg L -1 of Tween 80. After 24 hours of exposure, the live larvae were counted, considering those that did not move during observation or with the slight agitation of the vial.

Standardization of microbial inoculum for sensitivity tests
Two strains of bacteria were used: Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). These were previously identified and confirmed by biochemical tests.
Pure microbial cultures maintained in TSA agar were peaked for brain and heart infusion broth (BHI) and incubated at 35 °C until they reached exponential growth phase (4-6 h). After this period, the cultures had their cell density adjusted in 0.85% sterile saline solution, in order to obtain turbidity comparable to that of the standard McFarland solution 0.5, which results in a microbial suspension containing approximately 1.5 x 108 CFU mL -1 according to the standards of the Clinical and Laboratory Standards Institute (CLSI, 2020).

Disk Diffusion Method (DDM)
The disc diffusion technique was performed according to the Clinical and Laboratory Standards Institute (CLSI,2020), which standardizes the sensitivity tests of antimicrobials by disc-diffusion. First, the plates were prepared with the Culture Medium Mueller Hinton Agar (AMH) after its solidification was distributed to microbial suspension on the surface of the agar and left at room temperature for 30 min. Soon after the discs containing 50 μL of the EOs and the discs with defined concentrations of antibiotics are prepared. Using sterile tweezers, the discs were distributed on the surface of the agar. The plates were incubated in a bacteriological greenhouse at 35 °C for 24 hours. The diameters of the inhibition halos were measured, including the diameter of the disc. These trials were done in triplicate. The values of the inhibition halos were the mean measurements of the three results. Tests carried out in triplicate.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The Minimum Inhibitory Concentration (MIC) assay was performed using the broth dilution technique, proposed by the Clinical and Laboratory Standards Institute (CLSI,2020).
First, 2% solutions were prepared using dimethylsulfoxide (DMSO), and serial dilutions were prepared in MH Broth, resulting in concentrations of 10 to 1000 μg mL -1 . Microbial suspension containing 1.5 x108 CFU mL -1 of the E. coli and S. aureus strains was added to Research, Society and Development, v. 9, n. 7, e803974842, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4842 8 each concentration. The tubes were incubated at 35º for 24h. Sterility and growth controls were performed for the assay. After the incubation period, the MIC of the EO was verified, being defined as the lowest concentration that visibly inhibited bacterial growth (absence of visible cloudiness). Tests performed in triplicate.
For the Minimum Bactericidal Concentration (MBC) assay, an aliquot of 100 μL of the dilutions from MH broth that visibly inhibited microbial growth was used. The aliquots were inoculated in Mueller Hinton Agar (AMH) with subsequent incubation at 35°C for 24h.
The MBC was determined as the lowest dose that visually in the MIC assay showed growth inhibition and that in the culture in AMH also did not present bacterial growth.

Physicochemical properties
The physicochemical parameters of The EOs are important not only for quality determination, but also for the control of their purity and these are presented in Table 1. It is observed that the EO of C. sinensis obtained a yield of 2.47% higher than the EO of P. dioica of 1.80%. By individually comparing the yield of EO of C. sinensis to the results obtained by Silva et al., (2016) who extracted the EOs from the peel of dried and fresh fruits, the authors perceived their yield ranging from 1.80 to 2.00%, and this study obtained a yield of +0.47% above the maximum yield obtained by the authors, since the density of the same authors ranged from 0.8480 to 0.8490 g mL -1 , density that is similar to this work in a variation of +0.0010 g mL -1 .

Physicochemical parameters P. dioica C. sinensis
Density (g mL -1 ) 0,9820 0,8500 Even the EO yield of P. dioica being lower than the yield of C. sinensis it is important to emphasize that for EOs yields above 1.5% are of extreme significance. In a study conducted by Voris et al., (2017) when extracting this same EO from the fruit acquired in a retail market in Rio de Janeiro (RJ), the authors employed a period of 4 h in their hydrodistillation, but their maximum yield was 1.60%, compared to the current study that used a shorter hydrodistillation time (3h-100°C) and obtained a yield of 1.80% using a regenerative part of the plant , becomes of utmost importance and significance for visualizing its application potential.
Comparing the values for the EO studied with those of the literature, it can be observed that there was a similarity between them, with regard to the parameters analyzed.
The small differences in the values found can be attributed to factors such as collection time, different soil types, storage conditions and time (Costa et al., 2012). It is important to emphasize the yield of 2.47% for the EO of C. sinensis that was observed in results higher than the literature, encouraging its production due to the use of barks that are commonly discarded in public fairs or local neighborhoods of São Luís-MA.

Chemical characterization of essential oils
Chromatographic peaks were identified by comparing the respective mass spectra with data from the wiley 139 spectrothecae (1); (2) NIST107 and (3) NIST21. According to the results obtained, Table 2 presents the compounds identified in the EO extracted from the barks of C. sinensis and in Table 3 the compounds identified in the EO extracted from the leaves of P. dioica.As can be seen in Table 2, 15 components were identified in the EO sample of C. sinensis, being the majority constituent of EO d-limonene with 81.50% of the composition, followed by linalool (6.36%) and β-mircene (2.95%). Research, Society and Development, v. 9, n. 7, e803974842, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4842 D-limonene is a relatively stable terpene that has applications in the literature for the development of plant bioproducts (Granja et al., 2015). The EOs of the genus Citrus have this component as the majority in its composition and properties as antimicrobial activity can be proven by Rodrigues (2019), but when we portray C. sinensis its bactericidal potential has been little studied, and many studies have been reported in relation to its antimicrobial and larvicidal action (Rodríguez et al., 2017;Araújo et al., 2016). Thus, it is observed that the EO of C. sinensis has the potential to explore its bactericidal activity in this study, being of vital importance for the state and for the country a natural product obtained through the part of a vegetable that is commonly discarded or surface applications.

The chemical compound d-limonene is confirmed as the main constituent of EO by
As can be seen in Table 3, 07 components were identified in the sample, with the majority constituent of EO being eugenol with 85.673%, followed by chavicol (6.79%) and myrcene (2.76%).
Eugenol content (85.67%) reported in this study becomes significant when compared with Oliveira et al. (2009) who extracted the EO from the leaves of P. dioica collected in Minas Gerais also observed that eugenol as the major constituent, but the observed content was 44.9%. Another fact reported was the presence of limonene in 10.1% of the composition and the chavicol being exhibited in a content of 7.5%. This composition may also be linked to the lower yield of 0.49% obtained by Oliveira (2017), although it is emphasized that the Development, v. 9, n. 7, e803974842, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4842 authors used an extraction time of 4 h. consituinte of its EO sample of P. dioica leaves, but different from this secondary component study of the authors was myrcene with 8.19% and chavicol was followed by 6.35%.
Eugenol is an extraordinarily versatile molecule and has been included as a spicy aroma in ice cream, bakery products and sweets in restricted concentrations, mouthwashes, pharmaceutical and dental preparations (Oliveira et al., 2009;Padmakumari et al., 2011;Martinez-Velazquez et al., 2011). In addition to having biological properties proven by Kamatou et al., (2012), thus it is vitally important to study the EO extracted from P. dioica as a significant natural source of eugenol for both biological applications and industries in general. Table 4 presents the Lethal Concentrations 50% referring to the action of the EOs against Artemia salina L. and its subsequent classification according to the criterion Dolabela (1997). Lethal Concentration 50% (LC50) refers to the point where the number of surviving animals is equal to the number of dead animals, and following the dolabela criterion (1997) it is possible to determine the toxicity of natural products aiming at a specific application of the agent in the target organism, since oils with high toxicity are not recommended for biological applications. Table 4 shows that none of the oils were classified as toxic, so their applications can be relatively acceptable and encouraged. Thus, antimicrobial activity assays were initiated. It is important to highlight that the EO of C. sinensis extracted from fruit bark so far has a significant yield and chemical components of biological importance and in this toxicity assay presents the LC50 of 511.6 mg L -1 , well above the criterion that was only 250 mg L -1 to be classified as nontoxic. Therefore, this EO has its application potential again encouraged.

Toxicity
It is important to emphasize that studies related to the toxicity of natural products are of vital importance for biological applications and studies in the literature do not yet disclose toxicity of the plants under study in a specific test such as the bioassay against Artemia salina.

Antimicrobial activity
The results regarding the tests to determine antimicrobial activity are presented in Table 5. All oils showed antimicrobial activity against E. coli and S. aureus. Development, v. 9, n. 7, e803974842, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4842 13 Eldahshan & Halim (2016) still emphasize that this oil had this activity due to the presence of oxygenated compounds in its composition. The authors highlight the potential of EO to be used as antibacterial additives in food and cosmetic products in order to reduce dependence on synthetic food preservation chemicals (Eldahshan & Halim,2016). Finally, we highlight again the biological potential of both species studied in this work as extremely efficient in the control of pathogenic microorganisms, represented by E. coli as Gramnegative and S. aureus as Gram-positive.

Final Considerations
Through the results obtained in the chemical studies, in the evaluation of the toxicity and antimicrobial of The EOs of P. dioica and C. sinensis, it is concluded that the evaluated EOs are composed of substances that provide and encourage their application due to their potentials for antimicrobial biological activity.