Selina-1,3,7(11)-trien-8-one and Oxidoselina-1,3,7(11)-trien-8-one from Eugenia uniflora Leaf Essential Oil and Their Cytotoxic Effects on Human Cell Lines

The sesquiterpenes selina-1,3,7(11)-trien-8-one and oxidoselina-1,3,7(11)-trien-8-one were isolated from the essential oil of Eugenia uniflora L. leaves. The structures were elucidated using spectrometric methods (UV, GC–MS, NMR, and specific optical rotation). The relationship between antioxidant activity, as determined by DPPH assay, and the cytotoxic effect was evaluated using tumor cells, namely lung adenocarcinoma epithelial cells (A549) and human hepatoma carcinoma cells (HepG2), as well as a model of normal human lung fibroblast cells (IMR90). Both compounds did not show prominent free-radical scavenging activity according to DPPH assay, and did not inhibit lipid peroxidation in Wistar rat brain homogenate. The isolated compounds showed pro-oxidative effects and cytotoxicity in relation to the IMR90 cell line.


Introduction
Eugenia uniflora L. (Myrtaceae), popularly known in Brazil as pitangueira, is a small tree species widely distributed in South and Central America [1,2]. In view of the pharmacological activities attributed to the species in folk medicine, the plant has received intense scientific attention and become of interest to the cosmetic industry [3][4][5]. E. uniflora currently has at least 41 recognized and registered heterotypic synonyms in Brazil [6], which indicates the existence of a large number of varieties for this plant species.
Studies on the chemical composition of essential oils from E. uniflora leaves show that compounds 1 and 2 are not thermosensitive, but some other components can undergo Cope rearrangement transformations during gas chromatography analysis, a method that is proven to be especially inadequate for the analysis of sesquiterpenes such as furanodiene, curzerene, germacrene B, γ-elemene, germacrone, and β-elemenone [7,8]. This analytical aspect requires attention when interpreting the results of numerous publications on the chemical composition and biological activities of essential oils.
Several studies have shown the potential biological activities of E. uniflora essential oils. Costa (2010) demonstrated the antifungal activity of essential oils from E. uniflora leaves against Paracoccidioides brasiliensis for samples presenting curzerene, germacrene D, and germacrene A as the major GC constituents [10]. An essential oil from the leaves of E. uniflora, containing 1 and 2 as the major compounds, showed antifungal activity against standard strains of Candida albicans, C. krusei, and C. tropicalis [13]. Additionally, another essential oil from E. uniflora leaves presenting curzerene and high proportions of 1 and 2 according to GC analyses showed relevant hepatoprotective activity, and the therapeutic potential of both compounds for the development of herbal medicines was mentioned [14,15].
Studies on the chemical composition of essential oils from E. uniflora leaves show that compounds 1 and 2 are not thermosensitive, but some other components can undergo Cope rearrangement transformations during gas chromatography analysis, a method that is proven to be especially inadequate for the analysis of sesquiterpenes such as furanodiene, curzerene, germacrene B, γ-elemene, germacrone, and β-elemenone [7,8]. This analytical aspect requires attention when interpreting the results of numerous publications on the chemical composition and biological activities of essential oils.
Several studies have shown the potential biological activities of E. uniflora essential oils. Costa (2010) demonstrated the antifungal activity of essential oils from E. uniflora leaves against Paracoccidioides brasiliensis for samples presenting curzerene, germacrene D, and germacrene A as the major GC constituents [10]. An essential oil from the leaves of E. uniflora, containing 1 and 2 as the major compounds, showed antifungal activity against standard strains of Candida albicans, C. krusei, and C. tropicalis [13]. Additionally, another essential oil from E. uniflora leaves presenting curzerene and high proportions of 1 and 2 according to GC analyses showed relevant hepatoprotective activity, and the therapeutic potential of both compounds for the development of herbal medicines was mentioned [14,15].
Given the importance of volatile compounds as bioactive natural products from medicinal and food plants along with the need to track their different biological activities, two sesquiterpenes from the leaves of E. uniflora, selina-1,3,7-trien-8-one (1) and oxidoselina-1,3,7(11)-trien-8-one (2), were isolated using conventional column chromatography. Structural elucidation was performed using modern spectroscopy and specific optical rotation techniques. We also present, here, the results of antioxidant analyses for the two sesquiterpenes and an assessment of the relationship between antioxidant power and cytotoxic effects in different normal and cancerous human cells.

Results and Discussion
The chemical composition found in this work for the E. uniflora leaf oil is shown in Table 1. The sample of the essential oil studied here presents the striking profile of one of the best known chemotypes of this plant species [14][15][16][17][18], in which the four main components stand out, namely curzerene, germacrene B, and 1 and 2. In fact, the GC-MS analytical chromatogram of the essential oil obtained in the present work is even very similar to that obtained from leaves of the tree cultivated in Nigeria, which were used in a chemical study by Weyerstahl (1988) [8]. RRI a : values of calculated relative retention indices using the column Rtx-5MS (GC-MS) and the n-alkanes series C8-C19; RRI b : published relative retention indices for apolar columns [17,18].
Chemical transformations of several of the components of the E. uniflora leaf essential oil are known to be caused by the heating conditions commonly used in GC-MS analysis, and a cold method analysis is required for correct composition evaluation [7,8]. Particularly important for the case of the sample described in the present research are the transformations of germacrene B into γ-elemene, furanediene into curzerene, and germacrone into β-elemenone, noting that compounds 1 and 2 are stable under the heating conditions used in GC-MS.
Weyerstahl and collaborators (1988) first isolated and identified compounds 1 and 2 as the major components of the essential oil, and during the decades that followed, these sesquiterpenes were found to also occur in various E. uniflora chemotypes [8][9][10][11][12]. Compound 1 was isolated in our laboratory as an oil and presented optical activity [α] 20 D − 8 • (c 1.0, CHCl 3 ), very close to the value registered in the literature when the compound was isolated for the first time [8]. Compound 1, obtained by synthesis, presented the value of [α] 20 D − 258 • (c 1.0, CHCl 3 ) [19], which can be now considered as a very discrepant value when compared to that found for the natural substance.
Compounds 1 and 2 were evaluated for their antioxidant potential using the DPPH assay, and the results are shown in Figure 2. It is noteworthy that compounds 1 and 2 present insignificant free-radical scavenging activity. Regarding the inhibition of lipoperoxidation in rat brain homogenate, the compounds 1 and 2 did not show inhibitory activity at 40 mg/L. Thus, it is clear that compounds 1 and 2 do not present significant antioxidant activity through either single-electron transfer or hydrogen atom transfer (HAT) mechanisms. Garmus (2014) evaluated the antioxidant activity of the essential oil extracted from E. uniflora leaves and found that phenolic compounds found in the oil are mainly responsible for the antioxidant effect (determined using the DPPH assay). Similarly, Auricchio (2007) studied a hydroalcoholic extract from E. uniflora leaves and its inhibition of lipid oxidation in rat brain homogenate and found an IC 50 of 35 mg/L, which is different from the results obtained herein. Considering the data are obtained using two distinct mechanisms of antioxidant action, it is hypothesized that compounds 1 and 2 have no antioxidant potential [20,21].
Compounds 1 and 2 were evaluated for their antioxidant potential using the DPPH assay, and the results are shown in Figure 2. It is noteworthy that compounds 1 and 2 present insignificant free-radical scavenging activity. Regarding the inhibition of lipoperoxidation in rat brain homogenate, the compounds 1 and 2 did not show inhibitory activity at 40 mg/L. Thus, it is clear that compounds 1 and 2 do not present significant antioxidant activity through either single-electron transfer or hydrogen atom transfer (HAT) mechanisms. Garmus (2014) evaluated the antioxidant activity of the essential oil extracted from E. uniflora leaves and found that phenolic compounds found in the oil are mainly responsible for the antioxidant effect (determined using the DPPH assay). Similarly, Auricchio (2007) studied a hydroalcoholic extract from E. uniflora leaves and its inhibition of lipid oxidation in rat brain homogenate and found an IC50 of 35 mg/L, which is different from the results obtained herein. Considering the data are obtained using two distinct mechanisms of antioxidant action, it is hypothesized that compounds 1 and 2 have no antioxidant potential [20,21].  1 and 2 was higher than 500 µ M, indicating that it is necessary to use higher concentrations of these compounds to kill half of the cells. In the literature, it is recognized that phenolic compounds can act as either anti-or pro-oxidant agents [22] depending on their concentration, environmental pH, and the presence of metals and oxygen [23].  [12]. Compounds 1 and 2 promoted, most notably, antiproliferative effects on IMR90 normal cells (GI 50 = 184.7 and 14.9 µM, respectively) when compared with A549 (GI 50 = 590.8 and 359.3 µM, respectively) and HepG2 (GI 50 = 289.2 and 97.5 µM, respectively) cancer cells, indicating high cytotoxicity and low safety in in vitro studies. The lethal concentration (LC 50 ) for 1 and 2 was higher than 500 µM, indicating that it is necessary to use higher concentrations of these compounds to kill half of the cells. In the literature, it is recognized that phenolic compounds can act as either anti-or pro-oxidant agents [22] depending on their concentration, environmental pH, and the presence of metals and oxygen [23]. Herein, despite their antioxidant effect as pointed out by the DPPH assay, it was clear that both 1 and 2 compounds exerted pro-oxidant behavior (Figure 4) by inducing reactive oxygen species (ROS) generation in non-cancer (IMR90) and malignant (A549) cells, which explains their cytotoxicity observed in cell viability assay. It is known that if ROS levels increase dramatically to toxic concentrations, the JNK (c-Jun NH2-terminal kinases) pathway can be activated, resulting in apoptosis and cell death [22]. By contrast, Sobeh (2019) reported that the methanolic extract of E. uniflora leaves present noticeable antioxidant properties by reducing the intracellular ROS levels and by increasing the reduced glutathione (GSH) content in HaCaT cells [24]. This disagreement may be explained by the chemical profile of a crude extract compared to the isolated compounds 1 and 2. In this case, the antioxidant effect may have occurred due to the synergistic effects between the bioactive compounds the crude extract, while the oxidative activity shown herein was related to the individual abilities of compounds 1 and 2. Indeed, our results highlight the high cytotoxicity of compounds 1 and 2, acting in a more intense way against IMR90 normal cells than cancer cells (A549 and HepG2) because of their pro-oxidant behavior, as observed in the ROS generation assay.
Molecules 2021, 26, x FOR PEER REVIEW 7 of 11 Figure 4. Results for intracellular ROS in A549 and IMR90 cells treated with 1 and 2 (10, 50 and 100 μM) as measured by spectrofluorometry. The data were analyzed by one-way ANOVA and quantitative data are expressed by mean ± standard deviation.* Different letters in the columns mean statistical difference.
The antioxidant activity measured by chemical and biological assays, together with the cell-based antioxidant activity measurement, clearly indicates that 1 and 2 cannot be considered antioxidant agents. Data obtained using chemical antioxidant activity (inhibition of lipid peroxidation and DPPH assay) and the induced ROS generation show that 1 and 2 are not able to scavenge free radicals (DPPH, peroxyl and hydroxyl radicals) and decrease intracellular ROS generation. Thus, it is clear that any prospect of compounds 1 and 2 having antioxidant action is very limited. In addition, taken all together, results show pro-oxidative effects and cytotoxicity in relation to normal human cells. This poses a toxicological concern for both substances, and the data generated here can be used as a basis for other biological assays that may encompass antioxidant and cytotoxic studies of the compounds [25].

Essential Oil Extraction and GC-MS Analysis
Three portions of approximately 300 g of ground dried leaves were extracted for 2.5 h in a steam distillation glass distiller. The oil samples were pooled in ethyl ether, dried over anhydrous Na2SO4, filtered, and evaporated under vacuum and at low temperature, yielding 7.56 g (0.84% w/w) based on dry weight.
For the oil analyses by GC-MS (Shimadzu GCMS-QP2010 Plus Gas Chromatograph), a non-polar Rtx-5MS column (30 m × 0.25 mm × 0.25 μm) and the following analytical conditions were used: split ratio of 1/20, 250 °C for the injector, 250 °C for the ion source, and 280 °C for the interface. The oven temperature was programmed to 60 °C for the first 5 min, increasing at a rate of 3 °C/min to reach the final temperature of 240 °C. The components were identified based on the relative retention indices calculated using a series of Figure 4. Results for intracellular ROS in A549 and IMR90 cells treated with 1 and 2 (10, 50 and 100 µM) as measured by spectrofluorometry. The data were analyzed by one-way ANOVA and quantitative data are expressed by mean ± standard deviation. * Different letters in the columns mean statistical difference.
The antioxidant activity measured by chemical and biological assays, together with the cell-based antioxidant activity measurement, clearly indicates that 1 and 2 cannot be considered antioxidant agents. Data obtained using chemical antioxidant activity (inhibition of lipid peroxidation and DPPH assay) and the induced ROS generation show that 1 and 2 are not able to scavenge free radicals (DPPH, peroxyl and hydroxyl radicals) and decrease intracellular ROS generation. Thus, it is clear that any prospect of compounds 1 and 2 having antioxidant action is very limited. In addition, taken all together, results show pro-oxidative effects and cytotoxicity in relation to normal human cells. This poses a toxicological concern for both substances, and the data generated here can be used as a basis for other biological assays that may encompass antioxidant and cytotoxic studies of the compounds [25].

Essential Oil Extraction and GC-MS Analysis
Three portions of approximately 300 g of ground dried leaves were extracted for 2.5 h in a steam distillation glass distiller. The oil samples were pooled in ethyl ether, dried over anhydrous Na 2 SO 4 , filtered, and evaporated under vacuum and at low temperature, yielding 7.56 g (0.84% w/w) based on dry weight.
For the oil analyses by GC-MS (Shimadzu GCMS-QP2010 Plus Gas Chromatograph), a non-polar Rtx-5MS column (30 m × 0.25 mm × 0.25 µm) and the following analytical conditions were used: split ratio of 1/20, 250 • C for the injector, 250 • C for the ion source, and 280 • C for the interface. The oven temperature was programmed to 60 • C for the first 5 min, increasing at a rate of 3 • C/min to reach the final temperature of 240 • C. The components were identified based on the relative retention indices calculated using a series of n-alkanes (C8-C19) and the mass spectra from the apparatus database, followed by comparison with the published data [8,17,18].

Isolation and Structure Identification of Compounds
The essential oil sample (7.0 g) was fractionated on a hexane packed silica gel 60 (240-400 mesh, Merck) column chromatography eluted with hexane/ethyl acetate mixtures in a gradient of increasing polarity. The two main components of the oil were isolated in various fractions (1, 0.6 g; 2, 0.4 g) and presented as single well-defined spots in thin layer chromatography (Macharey-Nagel 60 G 254 0.2 mm plates) first observed under 254 nm UV light and then sprayed with H 2 SO 4 /MeOH (1:1) and heated on a hot plate.

Chemical and Biological Antioxidant Activities
The free-radical scavenging activity of 1 and 2 in relation to the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical was measured using the colorimetric method described by Brand-Williams (1995), and the results are expressed in mg of ascorbic acid equivalent per 100 g of material (AAE/100 g) [26]. To evaluate the capacity of hydrogen atom transfer (HAT) of the isolated compounds, male Wistar rat brain homogenate was used as substrate for the production of thiobarbituric acid reactive substances (TBARS) produced by Fe 2+induced oxidation undertaken at 37 • C, following the experimental conditions described elsewhere [27]. The inhibition of lipid peroxidation was expressed as % of inhibition. The animal protocol was approved by the Ethics Committee (UEPG, protocol 47/2017).

Cytotoxicity Assay
The in vitro cytotoxic effect of 1 and 2 compounds were analyzed in relation to A549 (lung adenocarcinoma epithelial cells-BCRJ code: 0033), HepG2 (human hepatoma carcinoma cells-BCRJ code: 0291), and IMR90 (human lung fibroblast-BCRJ code: 0118) cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich, COD-M5655) assay [28]. All cell lines were maintained in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (Sigma-Aldrich, COD-D8900), with added 10% fetal bovine serum (FBS, Gibco, COD-16000044) and 100 IU penicillin/100 µg streptomycin (Sigma-Aldrich, COD-P4333). The cell lines were grown in a humidified incubator at 37 • C containing 5% CO 2 . Briefly, the cells were seeded into 96-well plates at a density of 1 × 10 4 cells/well (HepG2 and A549), 5 × 10 3 cells/well (IMR90), 100 µL/well, and after 24 h, the cells were treated with 100 µL of serial concentrations (10-500 µM) of compounds 1 and 2 for 48 h. The stock solution of extracts contained 5% of dimethyl sulfoxide (DMSO) at a final concentration of 0.2% in cell medium for all in vitro assays. The IC 50 , GI 50 , and LC 50 parameters were performed in accordance with the method described by do Carmo (2018) [22], in which IC 50 is the concentration of the agent that inhibits growth by 50%, calculated as (T/C) × 100 = 50, where T = number of cells at time t of treatment; C = control cells at time t of treatment; GI 50 is the concentration of the agent that inhibits growth by 50% relative to untreated cells, calculated as ((T − T0)/(C − T0)) × 100 = 50, where T and C are the number of treated and control cells, respectively, at time t of treatment, and T > T0 where T0 is the number of cells at time zero; LC 50 is the concentration of the agent that results in a net loss of 50% cells relative to the number at the start of treatment, calculated as ((T − T0)/T0) × 100 = −50, where T < T0. The experiments were conducted in quadruplicate on three different days to assure the reproducibility of the generated data.

Intracellular Reactive Oxygen Species (ROS) Measurement
In order to assess the in vitro antioxidant/oxidant potential of 1 and 2, intracellular ROS generation was measured using a fluorescence technique as described by Carmo (2019) [29]. Briefly, A549 cancer cells (6 × 10 4 /well) and IMR90 normal cells (2 × 10 4 /well) were exposed to concentrations of 10, 50, and 100 µg/mL of 1 and 2 compounds, culture medium (negative control), and 15 µmol/L H 2 O 2 (positive control), for 1 h in DCFH-DA solution (25 mmol/L), at 37 • C and 5% CO 2 . Subsequently, cells were washed with PBS and a H 2 O 2 solution (15 µmol/L) added for recording of fluorescence. Intracellular fluorescence intensity of cells was measured at an excitation wavelength of 485 nm and at an emission wavelength of 538 nm [29]. The assays were performed in quadruplicate on three different days.

Statistical Analysis
The significance for intracellular ROS measurement was defined by one-way analysis of variance followed by Tukey's test. Analysis on sigmoidal dose-response for cytotoxicity was performed using nonlinear regression for curve fitting.

Conclusions
We obtained and confirmed the chemical structures of 1 and 2 here and also provide a simple method for their isolation. Our results for structure analyses and cytotoxicity confirm the importance of establishing more knowledge about the volatile metabolites of Eugenia uniflora.