Antitumor Effect of Guatteria olivacea R. E. Fr. (Annonaceae) Leaf Essential Oil in Liver Cancer

Guatteria olivacea R. E. Fries (synonym Guatteria punctata (Aubl.) R.A. Howard) is a tree of 10–27 m tall popularly known as “envira-bobó”, “envira-fofa”, “envireira”, “embira”, “embira-branca”, “embira-preta”, envira-branca”, and “envira-preta”, which can be found in the Brazilian Amazon biome. In this study, we evaluated the cytotoxic and antitumor effects of the essential oil (EO) obtained from the leaves of G. olivacea against liver cancer using HepG2 cells as a model. EO was obtained using a hydrodistillation Clevenger-type apparatus and was qualitatively and quantitatively characterized using GC–MS and GC–FID, respectively. The alamar blue assay was used to assess the cytotoxic potential of EO in a panel of human cancer cell lines and human non-cancerous cells. In HepG2 cells treated with EO, YO-PRO-1/propidium iodide staining, cell cycle distribution, and reactive oxygen species (ROS) were examined. In C.B-17 SCID mice with HepG2 cell xenografts, the efficacy of the EO (20 and 40 mg/kg) was tested in vivo. GC–MS and GC–FID analyses showed germacrene D (17.65%), 1-epi-cubenol (13.21%), caryophyllene oxide (12.03%), spathulenol (11.26%), (E)-caryophyllene (7.26%), bicyclogermacrene (5.87%), and δ-elemene (4.95%) as the major constituents of G. olivacea leaf EO. In vitro cytotoxicity of EO was observed, including anti-liver cancer action with an IC50 value of 30.82 μg/mL for HepG2 cells. In HepG2 cells, EO treatment increased apoptotic cells and DNA fragmentation, without changes in ROS levels. Furthermore, the EO inhibited tumor mass in vivo by 32.8–57.9%. These findings suggest that G. olivacea leaf EO has anti-liver cancer potential.


Introduction
Liver cancer is one of the deadliest cancers, with 905,677 newly diagnosed cases and 830,180 deaths worldwide in 2020 [1]. Current liver cancer therapy has serious side effects and tumor resistance, and it can prolong survival by just a few months [2]. Therefore, new therapies are needed.
The family Annonaceae has therapeutic potentials, in which the genus Guatteria Ruiz et Pav. is one of the most representative [3,4]. The antitumor potential of the essential oils (EO) from plants belonging to Guatteria has been previously explored, including

Chemical Composition of G. olivacea Leaf EO
The analysis by gas chromatography coupled with mass spectrometry (GC-MS) and flame ionization detector (GC-FID) made it possible to determine the chemical composition of the EO of G. olivacea from dry leaves obtained by hydrodistillation in triplicate. EO oil presented a light green coloration with a strong smell, and the yield obtained was 0.18 ± 0.02% in relation to the weight of the dry material of the samples in triplicate. The chemical compounds were identified based on their mass spectra (Figures S1-S13) and their respective retention rates, as well as a comparison with the data available in the literature. After the chemical analysis, a total of thirty-nine compounds were identified, which comprised 96.85% of the EO composition (Table 1). Among the identified compounds, only terpenoids were observed as constituents, from which sesquiterpenes (37 substances) were the dominant class, comprising 94.10% of the EO composition. Monoterpenes (two substances) were also observed but were represented by only 2.75% of the EO sample.
G. olivacea leaf EO showed IC 50 values less than 30 µg/mL for most cell lines tested and was considered promising according to our cytotoxic screening program for new compounds/extracts/oils [5,6,9,10,12,[23][24][25]. As a result, it was chosen for further research using the liver cancer HepG2 cells as a cellular model since liver cancer is one of the lethal cancers. These are the first data on the cytotoxicity of G. olivacea leaf EO. Interestingly, the EO of G. friesiana [5], G. pogonopus [6], G. blepharophylla [10], G. hispida [10], G. elliptica [11], and G. megalophylla [12] were also previously reported to have IC 50 values below 30 µg/mL against cancer cell lines. Cell death is a process that is triggered by a variety of factors and has distinct morphological characteristics depending on the type of cell death, such as apoptosis, necrosis, and autophagy, among others [26]. Apoptosis is the most type of cell death studied, in which this cell death pathway is the target of many cancer treatment strategies. Some features of apoptotic cell death include caspase activation, DNA fragmentation, phosphatidylserine externalization, loss of cell membrane integrity, and PARP cleavage [27]. In this context, the development of drugs with high apoptotic potential is critical.
The apoptotic cell death was quantified using YO-PRO-1/PI-staining HepG2 cells after treatment with EO at concentrations of 12.5, 25, and 50 µg/mL for 48 h. YO-PRO-1 is a nuclear marker that binds to the DNA of dying cells and emits a green fluorescence [28]. Its large size (630 Da) prevents it from penetrating the intact plasma membrane of living cells. However, apoptotic processes compromise membrane integrity, allowing YO-PRO-1 to enter cells, indicating that this dye is an early marker of apoptotic cell death. Its mechanism involves the activation of P2X7 receptors [28], while PI is a nuclear marker that binds to DNA only in dead or damaged cells and emits red fluorescence. As a result, when we use the combination of these two dyes, apoptotic cells show green fluorescence, dead cells (dead cells without identifying the type of cell death) show red and green fluorescence, and viable cells show little or no fluorescence.
As shown in Figure 2, a statistically significant increase in the percentage of cells undergoing apoptosis was found in HepG2 cells treated with EO at concentrations of 12.5 and 25 µg/mL after 48 h of treatment, while the increase in dead cells was observed at a concentration of 50 µg/mL. Doxorubicin, used as a positive control, significantly increased the percentage of dead cells. and 25 μg/mL after 48 h of treatment, while the increase in dead cells was observed at a concentration of 50 μg/mL. Doxorubicin, used as a positive control, significantly increased the percentage of dead cells. Furthermore, EO caused cell shrinkage, as measured by a decrease in forwarding light scatter, a morphological change seen in apoptotic cells ( Figure 3). Measurement of ROS levels was also performed after treatment of HepG2 cells with EO at the same concentrations for 1 and 3 h; however, no significant changes were observed (data not shown).  Negative control (NC) was treated with vehicle (0.5% DMSO) used for diluting EO, and doxorubicin (0.5 μg/mL) was used as positive control (PC). Data are presented as mean ± S.E.M. of at least three independent experiments. * p < 0.05 compared with negative control by ANOVA, followed by Student-Newman-Keuls test. Furthermore, EO caused cell shrinkage, as measured by a decrease in forwarding light scatter, a morphological change seen in apoptotic cells (Figure 3). Measurement of ROS levels was also performed after treatment of HepG2 cells with EO at the same concentrations for 1 and 3 h; however, no significant changes were observed (data not shown). and 25 μg/mL after 48 h of treatment, while the increase in dead cells was observed at a concentration of 50 μg/mL. Doxorubicin, used as a positive control, significantly increased the percentage of dead cells. Furthermore, EO caused cell shrinkage, as measured by a decrease in forwarding light scatter, a morphological change seen in apoptotic cells (Figure 3). Measurement of ROS levels was also performed after treatment of HepG2 cells with EO at the same concentrations for 1 and 3 h; however, no significant changes were observed (data not shown).  Negative control (NC) was treated with vehicle (0.5% DMSO) used for diluting EO, and doxorubicin (0.5 μg/mL) was used as positive control (PC). Data are presented as mean ± S.E.M. of at least three independent experiments. * p < 0.05 compared with negative control by ANOVA, followed by Student-Newman-Keuls test. Cell cycle phases and internucleosomal DNA fragmentation were also quantified in HepG2 cells treated with EO at concentrations of 12.5, 25, and 50 µg/mL for 48 h. A significant increase in cells with fragmented DNA was observed after treatment with EO at concentrations of 25 and 50 µg/mL ( Figure 4). Doxorubicin, used as a positive control, caused G 2 /M arrest and significantly increased cells with fragmented DNA.
Cell cycle phases and internucleosomal DNA fragmentation were also quantified in HepG2 cells treated with EO at concentrations of 12.5, 25, and 50 μg/mL for 48 h. A significant increase in cells with fragmented DNA was observed after treatment with EO at concentrations of 25 and 50 μg/mL ( Figure 4). Doxorubicin, used as a positive control, caused G2/M arrest and significantly increased cells with fragmented DNA. As mentioned above, the cytotoxicity of some EOs from the genus Guatteria has previously been reported in different histological types of cancer [5][6][7][8][9][10][11][12]. Among them, HepG2 cells treated with EOs obtained of G. blepharophylla and G. hispida caused cell morphologies consistent with apoptosis, as well as a remarkable activation of caspase-3 and DNA fragmentation without changes in cell membrane integrity [10]. Similarly, the EO of G. megalophylla increased phosphatidylserine externalization and DNA fragmentation in the leukemia HL-60 cells [12].
Notably, some of the major constituents of G. olivacea leaf EO have been reported to be cytotoxic. δ-Elemene, a sesquiterpene hydrocarbon, has been shown to induce cell death in human lung carcinoma cells by inhibiting the NF-kB pathway [29]. (E)-Caryophyllene, another sesquiterpene hydrocarbon, caused cytotoxicity in human lung cancer cells by causing G1 cell cycle arrest [30]. Germacrene D and bicyclogermacrene, other sesquiterpene hydrocarbons, have also been reported as cytotoxic agents against melanoma, leukemia, and colon cancer [31,32]. Caryophyllene oxide, a sesquiterpene oxygenated, caused apoptotic cell death in prostate cancer cells, as evidenced by depolarization of the mitochondrial membrane, morphological changes, and caspases activation [33]. Spathulenol, another sesquiterpene oxygenated, has been shown to be cytotoxic against melanoma, leukemia, and liver cancer cell lines [34].

In Vivo Antitumor Effect of G. olivacea Leaf EO
The antitumor activity of G. olivacea leaf EO was studied in CB-17 SCID mice inoculated with HepG2 cells. The animals were treated intraperitoneally with doses of 20 and 40 mg/kg of EO once a day for 21 days ( Figure 5). At the end of treatment, the mean weight of tumors in negative control animals was 0.90 ± 0.07 g. The mean tumor weights in EOtreated animals were 0.38 ± 0.14 and 0.61 ± 0.10 g at 40 and 20 mg/kg doses, respectively. Tumor mass inhibition was 57.9 and 32.8%, respectively. Doxorubicin reduced tumor As mentioned above, the cytotoxicity of some EOs from the genus Guatteria has previously been reported in different histological types of cancer [5][6][7][8][9][10][11][12]. Among them, HepG2 cells treated with EOs obtained of G. blepharophylla and G. hispida caused cell morphologies consistent with apoptosis, as well as a remarkable activation of caspase-3 and DNA fragmentation without changes in cell membrane integrity [10]. Similarly, the EO of G. megalophylla increased phosphatidylserine externalization and DNA fragmentation in the leukemia HL-60 cells [12].
Notably, some of the major constituents of G. olivacea leaf EO have been reported to be cytotoxic. δ-Elemene, a sesquiterpene hydrocarbon, has been shown to induce cell death in human lung carcinoma cells by inhibiting the NF-kB pathway [29]. (E)-Caryophyllene, another sesquiterpene hydrocarbon, caused cytotoxicity in human lung cancer cells by causing G 1 cell cycle arrest [30]. Germacrene D and bicyclogermacrene, other sesquiterpene hydrocarbons, have also been reported as cytotoxic agents against melanoma, leukemia, and colon cancer [31,32]. Caryophyllene oxide, a sesquiterpene oxygenated, caused apoptotic cell death in prostate cancer cells, as evidenced by depolarization of the mitochondrial membrane, morphological changes, and caspases activation [33]. Spathulenol, another sesquiterpene oxygenated, has been shown to be cytotoxic against melanoma, leukemia, and liver cancer cell lines [34].

In Vivo Antitumor Effect of G. olivacea Leaf EO
The antitumor activity of G. olivacea leaf EO was studied in CB-17 SCID mice inoculated with HepG2 cells. The animals were treated intraperitoneally with doses of 20 and 40 mg/kg of EO once a day for 21 days ( Figure 5). At the end of treatment, the mean weight of tumors in negative control animals was 0.90 ± 0.07 g. The mean tumor weights in EO-treated animals were 0.38 ± 0.14 and 0.61 ± 0.10 g at 40 and 20 mg/kg doses, respectively. Tumor mass inhibition was 57.9 and 32.8%, respectively. Doxorubicin reduced tumor mass by 25.6%. The H&Estained tumors showed a collection of proliferative hyperchromatic malignant cells exhibiting anisocytosis. Mitotic events were frequent in both groups, some atypical. In animals treated with EO at a dose of 40 mg/kg, tumor nodules were smaller and exhibited a denser surrounding connective tissue when compared to negative control. Necrotic areas were observed in all groups, especially in doxorubicin-treated mice. mass by 25.6%. The H&E-stained tumors showed a collection of proliferative hyperchromatic malignant cells exhibiting anisocytosis. Mitotic events were frequent in both groups, some atypical. In animals treated with EO at a dose of 40 mg/kg, tumor nodules were smaller and exhibited a denser surrounding connective tissue when compared to negative control. Necrotic areas were observed in all groups, especially in doxorubicin-treated mice. All groups had 100% survival rates, and no significant changes in body and organ weights (liver, kidney, lung, and heart) were observed in any group (p > 0.05) ( Figure 6). All groups had 100% survival rates, and no significant changes in body and organ weights (liver, kidney, lung, and heart) were observed in any group (p > 0.05) ( Figure 6).
Histopathological analyses of the liver, kidney, lung, and heart were also performed. The livers of all experimental groups presented a preserved portal architecture. In general, some morphological changes were observed, such as vascular congestion ranging from mild to moderate, mild hydropic degeneration, and coagulation necrosis in focal areas of the organ. Isolated inflammatory cells were observed around the portal system, predominantly polymorphonuclear cells. Importantly, only the animals treated with EO at dose of 40 mg/kg showed moderate microgoticular steatosis ( Figure S14). The renal architecture was preserved in all animals in the present study. However, some histopathological changes were observed, such as vascular congestion ranging from moderate to intense and a decrease in the urinary space (Bowman's space) by glomerular hyalinization. Focal areas of coagulation necrosis in the tubules of the renal cortex were observed in doxorubicinand EO-treated groups ( Figure S15). The architecture of the lung parenchyma ranged from preserved to partially preserved in this study. The histopathological changes observed were vascular congestion, edema, and thickening of the interalveolar septa with reduced alveolar lumen, which ranged from mild to intense. In addition, focal areas of hemorrhage, fibrosis, and inflammatory cells, predominantly polymorphonuclear, were observed in all experimental groups ( Figure S16). For the hearts, we did not observe any noteworthy architectural and morphological changes for this organ. Histopathological analyses of the liver, kidney, lung, and heart were also performed. The livers of all experimental groups presented a preserved portal architecture. In general, some morphological changes were observed, such as vascular congestion ranging from mild to moderate, mild hydropic degeneration, and coagulation necrosis in focal areas of the organ. Isolated inflammatory cells were observed around the portal system, predominantly polymorphonuclear cells. Importantly, only the animals treated with EO at dose of 40 mg/kg showed moderate microgoticular steatosis ( Figure S14). The renal architecture was preserved in all animals in the present study. However, some histopathological changes were observed, such as vascular congestion ranging from moderate to intense and a decrease in the urinary space (Bowman's space) by glomerular hyalinization. Focal Likewise, G. megalophylla leaf EO inhibited tumor mass by 16.63% at 50 mg/kg and 48.79% at 100 mg/kg in mice engrafted with HL-60 cells, with no significant change in the relative weight of organs from any of the groups studied [12]. The antitumor effect of G. friesiana leaf EO was studied in a murine model using S180-bearing mice. When administered intraperitoneally (50 and 100 mg/kg) and orally (100 and 200 mg/kg), the tumor growth inhibition rates were 43.4-54.2% and 6.6-42.8%, respectively [5]. G. pogonopus leaf EO also inhibited S180 tumor growth by 25.3-42.6% when administered intraperitoneally at 50 and 100 mg/kg, respectively [6].

Essential Oil Extraction
The leaves of G. olivacea were oven-dried with air circulation at 40 • C for 24 h and subjected to hydrodistillation (900 g) for 4 h using a Clevenger-type apparatus (Amitel, São Paulo, Brazil). Hydrodistillation was performed in triplicate (3 × 300 g = 900 g). The essential oil samples (in triplicate) were dried with anhydrous sodium sulfate (Na 2 SO 4 ), and the percentage of their content was calculated based on the weight of the dry material used in each hydrodistillation. Subsequently, the standard deviation of the triplicate was calculated. EOs were stored in the freezer prior to chemical and biological analyses.

GC-FID and GC-MS Analyses
The GC-FID analysis was carried out using a Shimadzu GC-17A GC system equipped with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Helium was the carrier gas at 1 mL/min flow rate. The injection solution was prepared by dissolving about 10 mg of oil in 1 mL of dichloromethane, being 1 µL of this solution injected at a split ratio of 1:50. The column temperature program was: 40 • C/4min, a rate of 4 • C/min to 240 • C, then a rate of 10 • C/min to 280 • C, and then 280 • C/2 min [35]. The injection and detector temperatures were 250 and 220 • C, respectively. The GC-MS analysis was carried out with a Trace Ultra gas chromatograph coupled to an ISQ single quadrupole mass spectrometer (Thermo Scientific). This system was equipped with a Tri Plus autosampler and a TR-5MS capillary column (30 m × 0.25 mm × 0.25 µm). The injection, interface, and ion source temperatures were 250, 250, and 220 • C, respectively. Mass spectrometry acquisitions were performed at a mass range of m/z 40-440. The other conditions were the same as those used for GC analysis.
The identifications of the oil components were performed based on a comparison of the obtained mass spectra with those stored in the NIST library and also by comparison of retention indexes (RI) with published data [22]. To obtain the RI, a homologous series of linear hydrocarbons (C 8 -C 20 ) was injected under the same analysis conditions, and the calculations were performed according to the Van den Dool and Kratz equation [21]. The standard ficoll density protocol was used to obtain primary cell culture of peripheral blood mononuclear cells (PBMC) from healthy donors' peripheral blood. PBMC were resuspended in RPMI 1640 medium containing 20% fetal bovine serum and 50 µg/mL gentamicin and maintained at 37 • C with 5% CO 2 . Concanavalin A (10 µg/mL, Sigma-Aldrich Co., Saint Luis, MO, USA) was added at the start of the culture as a mitogen to trigger cell division in T lymphocytes. The experimental protocol (number #031019/2013) was approved by the Oswaldo Cruz Foundation's Research Ethics Committee in Salvador, Bahia, Brazil.

Pharmacological Evaluation
The cell viability was determined using the Alamar blue assay, as previously described [36][37][38]. Cells were grown in 96-well plates for all experiments. EO was dissolved in dimethyl sulfoxide (DMSO, Vetec Qumica Fina Ltd.a, Duque de Caxias, RJ, Brazil) and added to each well before incubating for 72 h. Doxorubicin (doxorubicin hydrochloride, purity 95%, Laboratory IMA S.A.I.C., Buenos Aires, Argentina) was used as a positive control. At the end of the treatment, 20 µL of resazurin stock solution (0.312 mg/mL) (Sigma-Aldrich Co., Saint Luis, MO, USA) was added to each well. SpectraMax 190 Microplate Reader was used to measure absorbances at 570 and 600 nm (Molecular Devices, Sunnyvale, CA, USA).
Quantification of DNA content was used to assess internucleosomal DNA fragmentation and cell cycle distribution [40]. Cells were harvested in a permeabilization solution containing 0.1% triton X-100, 2 µg/mL PI, 0.1% sodium citrate, and 100 µg/mL RNAse (all from Sigma-Aldrich Co.), and flow cytometry was used to determine cell fluorescence.
At least 10 4 events were recorded per sample for all flow cytometry analyses. A BD LSRFortessa cytometer was used along with BD FACSDiva Software (BD Biosciences) and FlowJo Software 10 (FlowJo Lcc; Ashland, OR, USA). Cellular debris was omitted from the analysis.

Human Liver Cancer Xenograft Model
A total of 43 C.B-17 severe combined immunodeficient (SCID) mice (males and females, six weeks old, 20-25 g) were obtained and kept at the Gonçalo Moniz Institute-FIOCRUZ animal facilities (Salvador, Bahia, Brazil). The animals were kept in cages with free access to food and water. All animals were subjected to a 12:12 h light-dark cycle (lights on at 7:00 a.m.). The experimental protocol was approved by a local animal ethics committee (number #01/2021).
HepG2 cells (10 7 cells/500 µL/mouse) were implanted subcutaneously into the left front armpit, as previously described [23][24][25]. After one day, the animals were treated intraperitoneally (200 µL/mouse) once a day for 21 days. The mice were divided into four groups: #1 injected with vehicle (5% DMSO solution) used for diluting EO (n = 17); #2 injected with doxorubicin (0.8 mg/kg, n = 8); #3 injected with EO at 20 mg/kg (n = 9); and #4 injections with EO at 40 mg/kg (n = 9). One day after the end of treatment, an anesthetic overdose (thiopental, 100 mg/kg) was used to euthanize the animals, and tumors were excised and weighed. The inhibition ratio (percent) was calculated using the following formula: inhibition ratio (percent) = [(A − B)/A] × 100, where A is the average tumor weight of the negative control and B is the tumor weight of the treated group.
To assess toxicological effects, the mice were weighed at the start and end of the experiment. Throughout the study, animals were monitored for abnormalities. The tumor, livers, kidneys, lungs, and hearts were excised and weighed. Macroscopically, the organs were analyzed for the presence of metastatic nodules, color changes, and hemorrhage. Histological analyses for tumors and organs were performed under optical microscopy using hematoxylin-eosin and periodic acid-Schiff (liver and kidney) staining after fixation in 4% formaldehyde.

Statistical Analysis
The data were presented as mean ± S.E.M. or as the half-maximal inhibitory concentration (IC 50 ) value with 95% confidence intervals derived from nonlinear regressions from at least three independent experiments performed in duplicate. ANOVA was used to compare differences between experimental groups, followed by Student-Newman-Keuls test (p < 0.05). GraphPad Prism was used to perform all statistical analyses (Intuitive Software for Science; San Diego, CA, USA).