Comparative study of the chemical composition, larvicidal, antimicrobial and cytotoxic activities of volatile oils from E. punicifolia leaves from Minas Gerais and Goiás

Eugenia punicifolia (Kunth) D.C., Myrtaceae, known as “pedra-ume-caá”, is popularly used in the treatment of inflammation, infections, fever, flu, diabetes, and diarrhea. This study aimed to carry out a comparative study of the Research, Society and Development, v. 10, n. 11, e34101119354, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19354 2 chemical composition of volatile oil from E. punicifolia leaves collected in Goiás and Minas Gerais, as well as to evaluate the larvicidal activity against Aedes aegypti L3 larvae, the antimicrobial activity against bacteria, pathogenic fungi, and environmental, and cytotoxic activity to Balb 3T3 cells (murine fibroblasts). Volatile oils were obtained by hydrodistillation in a Clevenger apparatus and analyzed by Gas Chromatography Coupled to Mass Spectrometry (CG/MS). A total of 60 compounds were identified, the main components found in the leaves of Goiás being Germacrene D, bicyclogermacrene and β-longipenene and in the leaves collected in Minas Gerais they were (Z)caryophyllene, γ-cadinene, spathulenol, caryophyllene oxide, and α-cadinol. The larvicidal effect was moderate against Ae. aegypti, with LC50 of 85.53 μg / mL for samples from Goiás and LC50 of 91.52 μg / mL for samples from Minas Gerais. Both oils showed moderate bactericidal activity against K. rhiziphyla (ATCC 9341), M. luteus (ATCC 10240), and S. aureus (ATCC 29737). The oils from Goiás (IC50 706.7 μg / mL) and Minas Gerais (IC50 160.7 μg / mL) had a lower cytotoxic concentration than the toxic action for larvae and bacteria, evidencing a safety profile and an interesting therapeutic potential, mainly concerning to volatile oil from Goiás. Therefore, the volatile oils from E. punicifolia leaves collected in Goiás and Minas Gerais that presented moderate larvicidal activity for Ae. aegypti also presented a bactericide activity and less cytotoxicity against murine fibroblasts. This is the first study of the larvicidal, antimicrobial and cytotoxic activity of volatile oils from E. punicifolia leaves.


Introduction
Eugenia punicifolia (Kunth) DC, Myrtaceae, known as Pedra-ume-Caá, Myrtle, Red myrtle, Pitanga-do-campo, is found widely distributed in the Amazon, Cerrado, Caatinga, Atlantic Forest, and Pantanal (Sobral, et al., 2015). It is a shrub with a yellow cylindrical stem with light spots, the leaves are elliptical or opposite lanceolate and petiolate, the flowers are arranged in white panicles. It has ripe fruits that are simple, fleshy, with an intense red color with a glabrous and shiny surface, astringent, indehiscent flavor, with an obovate to an elliptical shape, with two or three seeds (Martins, 1989;Coneglian, 2007;Senra, 2012) Anatomically, it presents secretory cavities with diffuse distribution both on the leaf surface and on the petiole (Lemos, et al., 2019).
No studies on the larvicidal activity of E. punicifolia were found.
The aim of this study was to carry out a comparative study of the chemical composition of volatile oil from E. punicifolia leaves collected in Minas Gerais and Goiás, as well as to evaluate the larvicidal activity against L3 larvae of Aedes aegypti, antimicrobial activity against fungi, and bacteria (pathogenic and environmental), and cytotoxic activity to Balb 3T3 cells (murine fibroblasts). Research, Society andDevelopment, v. 10, n. 11, e34101119354, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19354

Plant material 2.1 Collect botanical material
The leaves of 10 individuals of Eugenia punicifolia (Kunth) D.C, Myrtaceae, were collected in the morning, in São Gonçalo do Abaté -Minas Gerias (MG) in September 2017, and in Hidrolândia -Goiás (GO) in October 2017. The species was identified by Prof. Dr. José Realino de Paula, an exsiccate was prepared and deposited in the Herbarium of the Federal University of Goiás under the number UFG-48579. The leaves were dried in a hot air oven at approximately 38º C.

Volatile oil extraction and GC-MS analysis
To extract the volatile oil, the dried botanical material (leaves) from the two regions was ground separately, immediately before each extraction, in a Poli® industrial blender (model LS-08MBR-N), submitted to hydrodistillation in a Clevenger-type apparatus for 3 hours. The volatile oils obtained were desiccated with Na2SO4, placed, and stored in a freezer at -22º C until use.
The volatile oils were submitted to chromatographic analysis, in the gas phase, coupled to mass spectrometry (CG/MS) in a Shimadzu GC-MS QP2010A apparatus, with a silica capillary column SBD-5 (30 m×0.25 mm ID, 0.25 m film thickness) (composed of 5% phenyl methylpolysiloxane) and programmed temperature as follows: 60-240 °C at 3 °C/min, then at 280 °C at 10 °C/min, ending with 10 min at 280 °C, with carrier gas with a flow rate of 1 ml/min and split mode at a ratio of 1:20 and the injection port set to 225°C. Operating parameters of the significant quadripolar mass spectrometer: interface temperature 240°C; Electron impact ionization at 70 eV with scan mass range 40-350 m/z at a sampling rate of 1 scan/s. The chemical constituents of the volatile oil were identified by comparing the mass spectra and retention indices with those reported in the literature for the most common components of volatile oils (Adams, 2007). Retention indices were calculated by co-injecting a mixture of hydrocarbons, C8 -C32, and using the Van Den Dool & Kratz equation (Dool & Kratz, 1963;Adams, 2007).

Larvicidal activity
The larvicidal tests were performed at the Insect Biology and Physiology Laboratory (IPTSP/UFG) in a biological chamber with a temperature of 25 °C ± 1 °C, relative humidity of 85% ± 5% and a 12-hour photophase (Silva et al., 2003). The tests were carried out in serially decreasing dilutions from 100 to 20 ppm, in 50 mL polystyrene containers containing 25 mL of solution and twenty L3 larvae of Ae. Aegypti and later, the mortality events were quantified after 24 hours of exposure, with all tests being performed in triplicate, and having water and surfactant with negative controls. To assess larvicidal activity, the following criteria were used: LC50 ≤ 50 μg / mL is considered active, between 50 and 100 μg / mL, moderate activity, between 100 and 750 μg / mL of efficient or effective activity, and LC50 ≤ 750 μg / mL, inactive (Komalamisra, et al., 2005, Neves, et al., 2017Silva, et al., 2021). The classification for antimicrobial activity criteria were MIC<100 μg/mL (good antimicrobial activity); MIC between 100-500 μg/mL (moderate antimicrobial activity); MIC between 500-1000 μg/mL (weak antimicrobial activity), and MIC above 1000 μg/mL (inactive) (Holetz, et al., 2002).

Cytotoxicity assay by neutral red dye incorporation method
Initially, Balb/c 3T3 cells (murine fibroblasts) were distributed in 96-well plates at a density of 3x10 3 cells/100µL/well, so that the peripheral wells were filled with culture medium, being cultured for 24 hours for adhesion. Then, the culture medium was removed from the plates and 50µl of complete medium was added to each well. Subsequently, the oils from the leaves collected in Goiás and Minas Gerais were prepared at a concentration twice the desired concentrations and added to the corresponding wells. In parallel, another plate was also prepared separately, in which the cells were exposed to 8 different concentrations of Sodium Lauryl Sulfate (SLS) (CASRN 151-21-3), covering the range from 100 to 6.8 µg/mL, as per described in protocol Nº. 129 of the OECD (2010).

Statistical analysis
The results were expressed as Mean ± Standard Deviation of the viability inherent to each concentration evaluated, and the mean of three independent experiments, carried out in sextuplicate, was analyzed. IC50 values were obtained through non-linear regression, and analyzes were conducted using the GraphPad Prism 5.0 software.

Larvicidal Activity
As for the toxicity of the volatile oil to Ae. aegypti in the third stage, it was possible to observe that the samples of volatile oils from the leaves collected in Goiás had moderate larvicidal activity against Aedes larvae, presenting an LC50 of 85.53 ± 5.32 μg/mL and LC90 of 129.60 ± 9 .85 µg/mL. The volatile oil from Minas Geais had a LC50 of 91.52 ± 6.24 μg/mL and a LC90 of 123.86 ± 10.88 μg/mL. Values were obtained through non-linear regression, with confidence intervals (α = 0.05).

Cytotoxicity assay
At the concentrations tested, volatile oils showed moderate cytotoxicity against normal fibroblasts (Balb/c 3T3) with IC50 160.7 µg/mL for volatile oil from Minas Gerais and IC50 706.7 µg/mL for Goiás compared to the positive control, Lauril Sodium Lauryl Sulfate (SLS), which is toxic with an IC50 of 37.08 µg/mL (Table 3 and 4).
According to the Guidance Document on Using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests (OECD, 2010), for the assay to be considered acceptable, at least one of the evaluated concentrations must present cytotoxicity > 0 % and ≤ 50% of cell viability. Oil from Goiás showed cell viability ≤ 50% at concentrations below 0.18 mg/mL and that from Minas Gerais at concentrations below 0.37 mg/mL. Given these parameters, it was verified that the dose-response curves (Table 4 and Figure 3) have values that are following the acceptability criteria.
Higher concentrations of β-caryophyllene may be related to water stress and collection time after 4 pm (Silva, et al., 2016). The production of caryophyllene oxide is related to the exposure of β-caryophyllene to atmospheric air after the extraction of volatile oil (Barros, et al., 2009). β-elemene and its isomers are by-products of germacrene and arise due to acidic and heating conditions at the time of extraction (Barros, et al., 2009). The hydrodistillation itself induces the formation of cadinol isomers due to the reaction of water with cadinyl cation, on the other hand, the exit of a proton from the cadynyl cation leads to the synthesis of α and γ-muurulene (Barros, et al., 2009;Steele, et al., 1998).
Volatile oil, after extraction, is prone to oxidative damage, chemical transformations, or polymerization, or isomerization reactions due to processing and storage conditions, temperature, light, and oxygen availability (Turek & Stintzing, 2013). The variability in yield and chemical composition of volatile oils from plants of the same species is intrinsically related to geographic location, ecological factors such as biome, plant health, soil type, available nutrients, water stress, predation and herbivory, genetic and physiological aspects (flowering, fruiting, reproductive rest and age) in addition to atmospheric parameters such as climate, temperature, insolation, and precipitation are related to the chemical diversity of volatile compounds, as well as seasonal aspects and harvest time (Gobbo-Neto & Lopes, 2007;Cruz, et al., 2014;Verma, et al., 2014).  (Magina, et al., 2009), Eugenia involucrata (MIC 875 µg/mL) for S. aureus (ATTCC 25923) (Toledo, et al., 2020). The oil from the stems, leaves, and flowers of E. chlorophylla showed moderate effect for K. rhizophila ATCC 9341 (MIC 500 µg/mL) and S.
The volatile oils of E. punicifolia from both regions showed moderate cytotoxicity against Balb/c 3T3 (normal fibroblasts) with IC50 706.7 µg/mL and IC50 of 160.7 µg/mL for oils from Goiás and Minas Gerais, respectively, compared to the positive control with IC50 37.08 µg/mL. There are no reports in the literature on the cytotoxic evaluation of volatile oil from Eugenia punicifolia, but Galeno, et al. (2014) found that the aqueous extract of E. punicifolia leaves showed low toxicity at concentrations of 50, 25 and 12 μg/mL for fibroblasts (3T3-L1 cell), compared to cells treated with doxorubicin at a concentration of 5 μg/ mL, and according to Costa, et al. (2016), the hydroalcoholic extract did not reduce the viability of human neutrophils (in vitro) at concentrations of 0.1-1000 ug/mL.
In the work by Sousa, et al. (2015), the oil from E. calycina leaves showed moderate cytotoxicity with CC50 137.4 μg/mL for HeLa cells, already in the work by Silva, et al. (2021) showed an IC50 of 266.8 for HeLa cells, and an IC50 of 312.1 µg/mL for Vero cells within 24 h of exposure. Some oils have a toxic effect on MRC5 (human fibroblast) cells, such as oil from the leaves of E. flavescens DC. (IC50 14.0 µg/mL), E. patrisii Vahl (IC50 18.1 µg/mL)  and E. uniflora (IC50 between 10.27 and 14.95 µg/mL) (Figueiredo, et al., 2019).

Conclusion
It was found as the major compounds of the volatile oil from E. punicifolia leaves collected in the Goiás the bicyclogermacrene, germacrene D, β-longipinene and δ-amorphene and collected from Minas Gerais, (Z)-caryophillene, αcadinol, γ-cadinene, and caryophyllene oxide. Both oils showed moderate activity to L3 larvae of Ae. aegypti, with LC50 of 85.53 and 91.52 μg/mL, for Goiás and Minas Gerias, respectively. The oils showed moderate activity against gram-positive bacteria, K. rhizophyla (ATCC 9341), M. luteus (ATCC 10240), and S. aureus (ATCC 29737), and moderate cytotoxicity in murine fibroblasts (Balb/c 3T3), thus these oils can be well tolerated against the biological system, however, further studies are needed to assess their toxicity in vivo. Although the oils have different concentrations of the majors and compounds, the biological activities evaluated in this study were similar, which suggests the synergistic action of the compounds in common.
This is the first study of larvicidal, antimicrobial, and cytotoxic activity of volatile oils from E. punicifolia leaves.