Essential oils of Eplingiella fruticosa populations: chemical, antioxidant, and cytotoxic analyses

This study investigates the variations in the chemical profiles and biological activities (antioxidant and cytotoxic) of Eplingiella fruticosa from the state of Sergipe, an endemic species from the Northeast region of Brazil. The essential oils were extracted from six populations by hydrodistillation and analyzed by GC/MS-FID. Cluster analysis was performed with the data of the constituents of the essential oils, and then a dissimilarity matrix, based on Euclidean distances, and a dendrogram, through the Ward clustering method, were constructed. The antioxidant activity of the essential oils was tested by different assays (DPPH, ABTS, β-carotene, and FRAP), and cytotoxic activity was tested by the SRB assay. The compounds found in greater amounts were α-pinene, β-pinene, 1,8-cineole, camphor, borneol, δ-elemene, α-cubebene, α-ylangene, (E)-caryophyllene, germacrene D, bicyclogermacrene, trans-calamenene, spathulenol, caryophyllene oxide, and viridiflorol. These compounds defined the formation of two groups. The first group was composed of the populations of São Cristóvão, Itaporanga, Japaratuba, and Malhada dos Bois municipalities and was characterized by the presence of the monoterpene camphor (8.39-11.27%) and B16F10 (64.94% cell viability) in Malhada dos Bois. The observations of this study may open a way to optimize the use of the E. fruticosa populations in relation to their cytotoxic properties.


Introduction
Plants have been an important source of treatments for illnesses for thousands of years. According to the World Health Organization (WHO), 80% of the world's population depends on folk medicine for their basic health needs. Awareness of traditional knowledge and of medicinal plants can play a fundamental role in the investigation and discovery of the natural resources of plants. This empirical knowledge helps the pharmaceutical industry to develop new medications that will be used in the treatment of various diseases (Sivakumar and Balasubramanian, 2020;Jamshidi-Kia et al., 2018).
Lamiaceae, with around 250 genera and more than 7,000 species distributed throughout the world, is among the largest plant families. This family is considered an important source of essential oils with analgesic, antioxidant, and anticancer properties (Mesquita et al., 2019;Uritu et al., 2018;Nieto, 2017). The genus Hyptis, the representative of the family with the largest number of species in Brazil, underwent taxonomic modifications after a recent review of the subtribe Hyptidinae, when a considerable part of its species were redistributed in eleven genera, including the genus Eplingiella (Harley and Pastore, 2012).
Eplingiella fruticosa, popularly known as "alecrim-de-vaqueiro", is a medicinal and aromatic plant distributed over the coast of the Northeast region of Brazil. The species is used in folk medicine for treating pain through infusion of its leaves.
Some studies have shown that its essential oil has anti-hyperalgesic effects and effects against neurodegenerative diseases (Oliveira Melo et al., 2020;Beserra-Filho et al., 2019).
Essential oils are mixtures of volatile compounds obtained from medicinal and aromatic plants that exhibit diverse activities, e.g., antioxidant activity, performed by chemical constituents that avoid oxidative damage and may act to prevent various degenerative diseases, including cancer (Jemli et al., 2016;Sousa et al., 2013). Cancer is an important health problem and one of the main causes of death throughout the world. In spite of advances, there are no effective drugs for curing most cancers. That requires the development of new drugs with high efficiency, low toxicity, low environmental impact, and low cost. There is interest in identifying metabolites of medicinal plants with the aim of developing anticancer herbal medicines or adjuvants for conventional therapies (Tariq et al., 2017;Bray et al., 2013). Therefore, the aim of this study was to characterize the essential oil of native populations of E. fruticosa found in the state of Sergipe, Brazil, and investigate its antioxidant and cytotoxic effect.

Extraction and identification of the chemical composition of the essential oils
The leaves collected were taken to the Department of Agronomic Engineering of the Federal University of Sergipe, where the material was dried in a forced air circulation laboratory oven at 40°C for five days. The essential oil was extracted by hydrodistillation in a modified Clevenger apparatus (Guenther, 1972). Samples of 70 g of dry leaves were distilled for 140 minutes (Ehlert et al., 2006). The essential oils were collected and stored in amber bottles at -20°C up to analysis of chemical composition.
The GC-MS and GC-FID data were simultaneously acquired using a detector separation system. The flow split ratio was 4:1 (MS:FID). A 0.62 m × 0.15 mm i.d. restriction tube (capillary column) was used to connect the splitter to the MS detector; and a 0.74 m × 0.22 mm i.d. restriction tube was used to connect the splitter to the FID detector. The injector temperature was 250°C and the temperature of the ion source was 200°C. The ions were generated at 70 eV at a scanning Research, Society andDevelopment, v. 10, n. 16, e341101623723, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23723 speed of 0.3 fragments (scans) s -1 detected in the interval of 40-350 Da. The FID temperature was adjusted to 250ºC, and the gases supplied to the FID were synthetic air, hydrogen, and helium at flow rates of 30, 300, and 30 mL.min -1 , respectively (Silva et al., 2018).
The constituents of the essential oils were identified based on comparison of the retention indices in the literature (Adams, 2007). For the retention index, the equation of Van Den Dool and Kratz (1963) was used in relation to a homologous series of n-alkanes (nC9 -nC18). Three equipment libraries, from WILEY8, NIST107, and NIST21, were also used, which allowed comparison of the spectra data with those contained in the libraries, using a similarity index of 80%.

Antioxidant activity
For evaluation of the antioxidant capacity of E. fruticosa, 1 µL.mL -1 stock solutions of essential oil were prepared, diluted in ethanol, and tested in different assays (Couto et al., 2019). The assays were conducted in the Food Science Laboratory of the Federal University of Sergipe.

DPPH (2,2-diphenyl-1-picrylhydrazyl)
The essential oils of E. fruticosa were evaluated by the DPPH radical scavenging assay adapted from Brand-Williams et al. (1995). Concentrations of 50 µL of sample were added to 150 µL of ethanol solution of DPPH at 6 × 10 -5 mol.L -1 , which were placed in microplates. The tests were accompanied by a control (reaction medium without the oil). After 30 minutes of rest in a dark environment, reduction of the DPPH radical was measured at a wavelength of 515 nm in a plate reader (Biotek, Synergy Mx). Reduction in the absorbance (Abs) values of the samples was correlated with that of the control, and the DPPH radical scavenging percentage was established, expressed by the following equation: scavenging % = [(Abscontrol -Abssample) / Abscontrol] x 100. The results of inhibition of the DPPH radical were expressed in the DPPH radical scavenging percentage.

ABTS [2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)]
For the assay regarding the antioxidant against the ABTS radical, the methodology described by Re et al. (1999), with adaptations, was used. The ABTS radical cation was prepared from the reaction of 5 mL of a 7 mM ABTS solution in 88 µL of a 140 mM potassium persulfate (K2S2O8) solution, incubated in the absence of light for 16 h. After that time, the ABTS solution was diluted in ethanol until reaching a solution with absorbance of 0.70 nm ± 0.05 nm at 734 nm. In a microplate, 30 µL of the sample was added to 300 µL of the ABTS radical solution. The determinations were accompanied by a control (reaction medium without oil). Reduction in the absorbance values of the samples was correlated with the absorbance of the control, and the ABTS radical scavenging percentage was established, expressed by the following equation: scavenging % = [(Abscontrol -Abssample) / Abscontrol] x 100. The results of ABTS radical inhibition were expressed in ABTS radical scavenging percentage.

β-carotene/linoleic acid
The β-carotene/linoleic acid co-oxidation method was performed according to the methodology described by Miller (1971), with some modifications. To prepare the emulsion, 10 μL of linoleic acid, 200 μL of β-carotene solution (2 mg.mL -1 ), and 50 μL of Tween 20 were dissolved in 500 μL of chloroform. The solution was placed in a gas exhaust hood until complete evaporation of the chloroform, and 25 mL of distilled water, previously saturated with oxygen for 30 min, was added. Then, 35 µL of the sample and 250 µL of the emulsifier solution were pipetted in the plate. The same experimental procedure was performed with ethyl alcohol as a control. Absorbance was read at 470 nm in a spectrophotometer at time 0 h, and then the plate was incubated in the dark at 50ºC for 2 h. After that procedure, a new reading was made at the same wavelength. The decay of optical density of the control (Absinitial -Absfinal) was considered as 100% oxidation. From this relation, the decrease in reading of absorbance of the samples was correlated with the control, thus establishing the percentage of oxidation protection of the samples by the following equation: % of oxidation protection = 100 -[(Abs sample x 100) / Abs control].

FRAP
For determination of the ferric reducing capacity of the essential oils of E. fruticosa, the methodology described by Singhal et al. (2014) was used. A 9 μL aliquot of the sample was pipetted in a microplate, and then 27 μL of distilled water and 270 μL of the FRAP reagent were added. The FRAP reagent is prepared shortly before the test, mixing 10 mL of 0.3 M acetate buffer, 10% (1 mL) of a 10 mM Tripyridyl-S-triazine (TPTZ) solution, and 1 mL (10%) of a 20 mM ferric chloride aqueous solution. The plate was incubated at 37ºC for 30 min and a reading was made at 595 nm. The same experimental procedure was performed with ethyl alcohol as a control. A ferrous sulfate (FeSO4) curve was prepared at concentrations from 125-1500 µM, exposed to the same conditions of the sample. The results were expressed in µM of equivalents of ferrous sulfate/mL.

Cytotoxicity
The assays were conducted in the Cancer and Leishmania Biology and Immunology Laboratory of the Federal University of Sergipe. The cell lines of lung cancer (A549), melanoma (B16F10), and glioma (C6) were acquired at the Federal University of Rio de Janeiro. The cells were cultivated in a humidified incubator at 37°C in 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic (10,000 U/mL penicillin, 10,000 mg/mL streptomycin).
The cytotoxic activity of the essential oils of E. fruticosa was determined by the Sulforhodamine B (SRB) assay, following a protocol adapted from Kasinski et al. (2015) and Orellana & Kasinski (2016). The cell lines A549, B16F10, and C6 were sown in 200 μL of medium (10 4 cells/ml) in 96-well cell culture plates. After 24 h, the cells were treated with the oils at a concentration of 50 μM for 72 h. Dimethyl sulfoxide (DMSO, 0.05%) was used as a vehicle control and 10 μM Doxorubicin Hydrochloride was used as a positive control.
After the treatment, the supernatant was discarded and the cells were fixed with 100 μL of 30% trichloroacetic acid for 1 h at 4°C and stained with 100 μL of SRB (0.057% w/v) for 30 min. The plate was washed with 1% acetic acid three times to remove excess stain, and the intracellular stain was dissolved in 200 μL of TRIS (10 mM; pH 10.5) for 30 min. Absorbance was read in a microplate reader at 510 nm. The absorbance was converted into growth inhibition by the following equation:

Statistical analysis
Analysis of variance (ANOVA version 5.6) was performed on the data of chemical characterization, and the mean values were compared by the Scott-Knott test (p ≤ 0.05) (Ferreira, 2011). From the data of the constituents of the essential oils, two multivariate analyses were performed, principal component analysis and cluster analysis, using the Statistica® software. ANOVA was performed on the data of the antioxidant and cytotoxicity activity analyses (Ferreira, 2011). The data were expressed in mean ± SE (standard error) and Tukey's test (p < 0.05). All the tests were performed in triplicate.

Results
The concentrations of the main compounds of the essential oil varied significantly among the populations of E. fruticosa (Table 1). Among the populations, 51 compounds were found, and the following compounds were detected in greater quantities: α-pinene, β-pinene, 1,8-cineole, camphor, borneol, δ-elemene, α-cubebene, α-ylangene, (E)-caryophyllene, germacrene D, bicyclogermacrene, trans-calamenene, spathulenol, caryophyllene oxide, and viridiflorol. These compounds defined the formation of two groups (Figure 1). The first group was formed by the populations of São Cristóvão, Itaporanga d'Ajuda, Japaratuba, and Malhada dos Bois and was characterized by the presence of the monoterpene camphor (8.39-11.27%) as the compost of greatest concentration in relation to the other municipalities ( Figure 2). The second group was constituted by the populations of Moita Bonita and Pirambu and was characterized by the major presence of the sesquiterpene bicyclogermacrene (7.45% and 10.98%) (Figure 2).
According to principal component analysis (Figure 3), the first principal component represented 60.95% of the total variation and was positively related to the compounds α-pinene, β-pinene, 1,8-cineole, trans-calamenene, and caryophyllene oxide; and negatively correlated with δ-elemene, (E)-caryophyllene, germacrene D, and bicyclogermacrene. The second principal component represented 17.22% of the total variation and was positively related to the compounds α-cubebene and viridiflorol, and negatively correlated with the compounds camphor and borneol.  (Adams, 2017). Source: Authors

Antioxidant capacity of the essential oils of E. fruticosa
The results of antioxidant activity (DPPH, ABTS, β-carotene, and FRAP) were reported in Table 2. The sample from Itaporanga d'Ajuda exhibited the greatest radical scavenging activity (6.96%) in the DPPH test. In contrast, for the ABTS + radical cation test, the populations of Moita Bonita (3.84%), Japaratuba (3.40%), and Pirambu (3.29%) exhibited activity superior to that of the others.
In the oxidation protection test with β-carotene, the populations of Pirambu, São Cristóvão, and Malhada dos Bois showed greater capacities of inhibiting lipid peroxidation, with 10.16%, 9.50%, and 9.27%, respectively. The capacity for inducing reduction in the iron ion, measured with the FRAP test, was greatest for the sample from Malhada dos Bois, with a value of 206.00 µM/mL (Table 2). Mean values ± standard deviation of at least three independent experiments after treatments with the essential oil of E. fruticosa. Mean values followed by the same letter in the columns do not differ from each other by Tukey's test (p ≤ 0.05).

Cytotoxicity effect of the essential oils of E. fruticosa populations
The essential oils of E. fruticosa (Figure 4) were evaluated regarding their effect on reduction in cell growth of three lines of tumor cells, A549 (lung cancer), B16F10 (melanoma), and C6 (glioma). Doxorubicin (positive control) showed high growth inhibition in the three tumor lines evaluated (87.14 -94.63%). Among the essential oils, the Japaratuba population exhibited greater cytotoxicity in relation to the others for the line A549, with cell inhibition of 51.00%. In B16F10, the highest degree of inhibition (64.94%) was observed for the essential oil from the Malhada dos Bois population. There was no difference in C6, and the degree of inhibition ranged from 30.62% (Malhada dos Bois) to 49.40% (Japaratuba) (Figure 4).

Discussion
The essential oils of E. fruticosa are characterized by the variability in their components (Silva et al., 2018). This chemical polymorphism results from diverse factors, including genetic and environmental effects that, in combination or independently, affect their composition and quantitative and qualitative properties. The locations where the alecrim-devaqueiro populations were collected have similar topographic, soil, and mean annual temperature characteristics (Silva et al., 2018). Recently, an ecogeographic study of E. fruticosa described a common growth environment of the species, with sandy soils of low fertility and high aluminum (Al +3 ) concentration and content (Oliveira et al., 2021). The small variation observed in the composition of the essential oils among the E. fruticosa populations can be partially attributed to the low phenotypic plasticity of the plants to adapt to different environmental conditions. The limited geographic distribution of this medicinal plant may be a significant explanation of its chemical stability (Silva et al., 2018). Thus, it produces what is necessary for adaptation to the environment in which it occurs. If the environment does not vary, it does not suffer "pressure" to produce other compounds in response to the environment.
In the present study, the terpenoids camphor and bicyclogermacrene were the most representative compounds among the groups formed with the populations. Oliveira Melo et al. (2020) identified 26 compounds in the essential oil of E. fruticosa; the sesquiterpenes (65.7%), monoterpenes (33.23%), and the principal components included (E)-caryophyllene (14.16%), bicyclogermacrene (12.68%), 1,8-cineole (11.03%), α-pinene (6.79%), and β-pinene (5.10%). The compounds 1,8-cineole, spathulenol, α-pinene, and β-pinene were the principal components found in another study analyzing the composition and variability of the essential oil of E. fruticosa (Franco et al., 2011). The monoterpenoids (C10) and the sesquiterpenoids (C15) are widely distributed in the plants and are the most common constituents of volatile compounds in flowers, fruit, stems, and leaves. In addition to playing important roles in plant-environment interactions, many of them also have high commercial value because of their diverse biological activities (Anulika et al., 2016).
All the populations in the study exhibited low antioxidant abilities in the different tests. The antioxidant activity of an essential oil depends on its chemical composition. A good antioxidant response can be expected from essential oils with a large phenolic content and a modest unsaturated terpene content (Amorati et al., 2013;Miguel, 2010). Phenolic compounds have redox properties and therefore play an important role in the neutralization of free radicals (Dhifi et al., 2016). Therefore, the weak antioxidant activity of the oils of E. fruticosa may be attributable to their not having phenolic compounds in their composition. In a study with essential oils of Ocimum kilimandscharicum populations, greater antioxidant activity was identified in the plants with the presence of phenylpropanoids (eugenol, methyl chavicol) compared to populations predominated by terpenes, such as camphor (Chaturvedi et al., 2018). The same characteristic was observed in Ocimum basilicum, where the cultivars with the best antioxidant performance were characterized by those with essential oils rich in eugenol (Couto et al., 2019). This is the first study regarding the antioxidant and cytotoxicity activities of the essential oil of E. fruticosa. The populations from Japaratuba and Malhada dos Bois stood out with the highest degrees of inhibition in A549 and B16F10, respectively. Their oils are found in Group 1, in which camphor stood out as the compound with the highest concentration in relation to the populations of Group 2. Currently, the anticancer activity of various natural compounds derived from plants, such as essential oils, has been tested (Kaushik;DeBerardinis, 2018). Essential oils of Chiliadenus antiatlanticus, which have camphor as the major component in their constitution, showed significant toxicity for liver tumor cell lines HepG2 (55.8% cell inhibition) and melanoma B16 4A5 (41.6% cell inhibition) at 100 μg/mL (El Yaagoubi et al., 2021). Coté et al. (2017) evaluated the cytotoxic activity of the essential oil of Tanacetum vulgare and its principal components and identified the cytotoxic effect against A549 lung cancer with concentrations greater than 200 µg/mL of the essential oil, whereas the main constituents of camphor, borneol, and 1,8-cineole were all inactive at concentrations greater than 200 µg/mL. The mechanisms involved in antiproliferative activity mediated by essential oils include a halt in the cell cycle, apoptosis, and DNA repair mechanisms. The essential oils also act in reducing tumors, inhibiting metastases, and as antimultiresistance to medication molecules (Mesquita et al., 2019). Nevertheless, the great variation in the chemical profile of the essential oils means great diversity in the mechanisms of action and molecular targets. In addition, as these oils consist of a wide variety of compounds, each one of them can modulate or alter the effects of the others.

Conclusion
As a result in this study, little variation was observed in the composition of the essential oils among the E. fruticosa populations. Fifty-one compounds were found; α-pinene, β-pinene, 1,8-cineole, camphor, borneol, δ-elemene, α-cubebene, αylangene, (E)-caryophyllene, germacrene D, bicyclogermacrene, trans-calamenene, spathulenol, caryophyllene oxide, and viridiflorol were the main metabolites. The plants showed weak effects in terms of antioxidant activity; however, the populations of Japaratuba and Malhada dos Bois exhibited moderate cytotoxicity for cell lines of A549 and B16F10, which may be related to the presence of the monoterpene camphor. More studies are needed to elucidate the role those different components of the essential oil of E. fruticosa play in cytotoxicity for cell lines alone or in combination.