Antitumor Effect of the Essential Oil from the Leaves of Croton matourensis Aubl. (Euphorbiaceae)

Croton matourensis Aubl. (synonym Croton lanjouwensis Jabl.), popularly known as “orelha de burro”, “maravuvuia”, and/or “sangrad’água”, is a medicinal plant used in Brazilian folk medicine as a depurative and in the treatment of infections, fractures, and colds. In this work, we investigated the chemical composition and in vitro cytotoxic and in vivo antitumor effects of the essential oil (EO) from the leaves of C. matourensis collected from the Amazon rainforest. The EO was obtained by hydrodistillation using a Clevenger-type apparatus and characterized qualitatively and quantitatively by gas chromatography coupled to mass spectrometry (GC–MS) and gas chromatography with flame ionization detection (GC–FID), respectively. In vitro cytotoxicity of the EO was assessed in cancer cell lines (MCF-7, HCT116, HepG2, and HL-60) and the non-cancer cell line (MRC-5) using the Alamar blue assay. Furthermore, annexin V-FITC/PI staining and the cell cycle distribution were evaluated with EO-treated HepG2 cells by flow cytometry. In vivo efficacy of the EO (40 and 80 mg/kg/day) was demonstrated in C.B-17 severe combined immunodeficient (SCID) mice with HepG2 cell xenografts. The EO included β-caryophyllene, thunbergol, cembrene, p-cymene, and β-elemene as major constituents. The EO exhibited promising cytotoxicity and was able to cause phosphatidylserine externalization and DNA fragmentation without loss of the cell membrane integrity in HepG2 cells. In vivo tumor mass inhibition rates of the EO were 34.6% to 55.9%. Altogether, these data indicate the anticancer potential effect of C. matourensis.


Introduction
The genus Croton (family Euphorbiaceae) contains approximately 1300 species that are found in tropical and subtropical regions of the world [1]. Plants from this genus have been used in the folk medicine to treat cancer, which include C. palanostigma Klotzsch [2,3], C. lechleri Müll. Arg. [4] and C. tiglium L. [5]. Moreover, numerous plants belonging to this genus have been reported with cytotoxic
Then, we quantified the cell viability using annexin V-FITC and PI double staining, and the cell fluorescence was measured by flow cytometry to determine the percentage of HepG2 cells in viable (cells that are annexin V-FITC/PI double-negative), early apoptotic (cells that are annexin V-FITC-positive and PI-negative), late apoptotic (cells that are annexin V-FITC/PI double-positive), and necrotic (cells that are annexin V-FITC-negative and PI-positive) stages after 48 h of treatment with the EO at concentrations of 12.5, 25, and 50 µg/mL, as shown in Figure 1. These concentrations were based at the IC 50 value (28.5 µg/mL) of the EO for HepG2 cells, and since the doubling time of HepG2 cell is about 48 h, we used this time of treatment to evaluate the effect of the EO after one full cell cycle. The EO treatment caused an increase of the apoptotic (early + late apoptotic cells) cell death after 48 h of treatment (P < 0.05). No increase in the necrotic (annexin V-FITC-negative/PI-positive) cells was observed (P > 0.05). Doxorubicin also led to an increase of the apoptotic cells (P < 0.05). At the concentrations of 12.5, 25, and 50 µg/mL, the EO increased the apoptotic cell death to 12.1%, 23.6%, and 25.7%, against 6.3% observed at the control group. Doxorubicin, at 1 µg/mL, increased the apoptosis to 20.7%. Then, we quantified the cell viability using annexin V-FITC and PI double staining, and the cell fluorescence was measured by flow cytometry to determine the percentage of HepG2 cells in viable (cells that are annexin V-FITC/PI double-negative), early apoptotic (cells that are annexin V-FITC-positive and PI-negative), late apoptotic (cells that are annexin V-FITC/PI double-positive), and necrotic (cells that are annexin V-FITC-negative and PI-positive) stages after 48 h of treatment with the EO at concentrations of 12.5, 25, and 50 µg/mL, as shown in Figure 1. These concentrations were based at the IC50 value (28.5 µg/mL) of the EO for HepG2 cells, and since the doubling time of HepG2 cell is about 48 h, we used this time of treatment to evaluate the effect of the EO after one full cell cycle. The EO treatment caused an increase of the apoptotic (early + late apoptotic cells) cell death after 48 h of treatment (P < 0.05). No increase in the necrotic (annexin V-FITC-negative/PI-positive) cells was observed (P > 0.05). Doxorubicin also led to an increase of the apoptotic cells (P < 0.05). At the concentrations of 12.5, 25, and 50 µg/mL, the EO increased the apoptotic cell death to 12.1%, 23.6%, and 25.7%, against 6.3% observed at the control group. Doxorubicin, at 1 µg/mL, increased the apoptosis to 20.7%.  Data are presented as the means ± SEM. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris was omitted from the analysis. * P < 0.05 compared with the negative control by ANOVA, followed by Bonferroni's multiple comparison test.
The cell cycle distribution in the EO-treated HepG2 cells was performed by the DNA content using flow cytometry after 48 h of treatment, as shown in Figure 2. All DNA that was sub-diploid in size (sub-G 0 /G 1 ) was considered fragmented. EO-treated HepG2 cells presented an internucleosomal DNA fragmentation significantly increased (P < 0.05). Doxorubicin also significantly induced internucleosomal DNA fragmentation (P < 0.05). At the concentrations of 12.5, 25, and 50 µg/mL, the EO increased the DNA fragmentation to 10.0%, 13.2%, and 40.5%, against 7.0% observed at the control group. Doxorubicin, at 1 µg/mL, increased the DNA fragmentation to 22.4%. The cell cycle phases G 0 /G 1 , S, and G 2 /M were reduced proportionally. Data are presented as the means ± SEM. of three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris was omitted from the analysis. * P < 0.05 compared with the negative control by ANOVA, followed by Bonferroni's multiple comparison test.

In Vivo Antitumor Activity
In vivo antitumor effect of the EO from the leaves of C. matourensis was assessed in C.B-17 severe combined immunodeficient (SCID) mice with HepG2 cell xenografts. Animals were treated with the EO at doses of 40 and 80 mg/kg/day through intraperitoneal injections delivered once a day for 21 consecutive days. At the end of the treatment, the mean tumor mass weight of the negative control animals was 0.5 ± 0.1 g, as shown in Figure 3A. In the EO-treated animals, the mean tumor mass weights were 0.3 ± 0.1 g at 40 mg/kg/day dosage and 0.2 ± 0.03 g at 80 mg/kg/day dosage. Tumor mass inhibition rates of the EO were 34.6% to 55.9% (P < 0.05), as shown in Figure 3B. In this study, 5-fluorouracil at the dosage of 10 mg/kg/day was used as the positive control, which reduced the tumor weight by 44.2%. mass weights were 0.3 ± 0.1 g at 40 mg/kg/day dosage and 0.2 ± 0.03 g at 80 mg/kg/day dosage. Tumor mass inhibition rates of the EO were 34.6% to 55.9% (P < 0.05), as shown in Figure 3B. In this study, 5-fluorouracil at the dosage of 10 mg/kg/day was used as the positive control, which reduced the tumor weight by 44.2%.   In regard to the toxicological parameters investigated, no significant changes on body and organs (liver, kidney, lung, and heart) weight were seen on the EO-treated groups (P > 0.05), as shown in Table 3. Hematological parameters of the peripheral blood drawn from C.B-17 SCID mice with HepG2 cell xenografts were also analyzed, as shown in Table 4. Although some variations have been observed, no statistically significant alteration was found in the hematological parameters in the animals treated with the EO (P > 0.05). Table 3. Effect of the essential oil (EO) from the leaves of Croton matourensis on body and relative organ weight from C.B-17 SCID mice with HepG2 cell xenografts.

Discussion
In this study, antitumor effect of the EO from the leaves of C. matourensis was reported for the first time at this communication. As mentioned above, a previous study investigated the chemical composition and cytotoxic activity of the EO from the leaves of C. matourensis collected in Venezuela [19]. These authors found fenchyl acetate, methyleugenol, isoelemicine, elemicine, spathulenol, and valencene as the main constituents of this EO. Moreover, previous studies with the EO from the leaves, fruit, inflorescence, and bark of C. matourensis collected from the Brazilian Amazon rainforest presented α-pinene, elemecine, α-phellandrene, p-cymene, linalool, and β-caryophyllene as the main constituents [16,17]. Herein, we observed a different chemical composition of the EO for C. matourensis in comparison with individuals collected in Venezuela, where β-caryophyllene, thunbergol, cembrene, p-cymene, and β-elemene were found as major constituents. Interestingly, when compared to with a specimen collected in central Amazonia, especially, in Brazil, a similar chemical composition is observed.
In the work of Compagnone et al. [19], the EO from the leaves of C. matourensis caused cytotoxicity in LoVo (human colon carcinoma) and HeLa (human cervical cancer) cell lines with IC 50 values of 36.6 and 83.9 µg/mL, respectively, while inducing cytotoxicity in human dermis fibroblasts cells with an IC 50 value of 132.7 µg/mL. In this work, we observed similar results where the EO was cytotoxic with IC 50 values of 23.3 µg/mL for MCF-7, 28.9 µg/mL for HCT116, 28.5 µg/mL for HepG2, 17.8 µg/mL for HL-60, and 25.8 µg/mL for MRC-5.
In our cytotoxic screening program, EO with IC 50 values below 30 µg/mL are considered promising for cancer drug development [20][21][22]. Therefore, the results obtained with the EO from the leaves of C. matourensis should be explored further. These data corroborate with previous results observed with EO obtained from Croton species. The EO from the leaves of C. flavens showed cytotoxicity in A-549 (human lung carcinoma) and DLD-1 (human colon adenocarcinoma) cells with IC 50 values of 27 and 28 µg/mL, respectively [23]. The EO from the leaves of C. regelianus induced cytotoxic effect in HL-60, HCT-8 (human colon carcinoma), SF295 (human glioblastoma), MDA-MB-435 (human melanoma) cells, with IC 50 values of 22.2, 40.0, 48.0, and 47.3 µg/mL, respectively [6]. The EO obtained from leaves of C. campestres exhibited cytotoxicity in MCF-7 and HT-29 (human colon adenocarcinoma) cells with IC 50 values of 8.61 and 9.94 µg/mL, respectively [24].
Concerning the main constituents of the EO from the leaves of C. matourensis, some of them, e.g., β-caryophyllene, cembrene, p-cymene, and β-elemene, have been reported as cytotoxic agents [22,[25][26][27][28], indicating that the mixture of them are associated with the cytotoxic and antitumor properties of this EO.
Phosphatidylserine externalization and DNA fragmentation, without loss of the cell membrane integrity, are cellular biochemical alterations that are often correlated with the apoptotic cell death [29]. Herein, we observed that the EO from the leaves of C. matourensis increased the phosphatidylserine exposure and internucleosomal DNA, without causing alterations in the cell membrane integrity (no increase in the PI-positive cells was observed). Moreover, the EO from the leaves of Lippia gracilis, Guatteria blepharophylla, and Guatteria hispida inhibited cell proliferation and induced cell death by apoptosis in HepG2 cells [25,30]. The EO from the aerial parts of Salvia aurea, S. judaica, and S. viscosa caused the apoptosis process and increasing reactive oxygen species in DU-145 (human prostate cancer) cells [31]. The EO from the leaves of Pinus roxburghii induced apoptosis along with inhibition of NF-κB and inhibited the expression of genes associated to cell survival (survivin, c-FLIP, Bcl-2, Bcl-xL, c-Myc, c-IAP2), proliferation (cyclin D1), and metastasis (MMP-9) in KBM-5 (chronic myeloid leukemia) cells [32].
In addition, we demonstrated that the EO from the leaves of C. matourensis has in vivo antitumor effect in C.B-17 SCID mice with HepG2 cell xenografts. The antitumor effect of the EO from the leaves of C. regelianus has been previously reported using sarcoma 180 as tumor model, while the EO inhibited the tumor development by 28.1% and 31.8%, at doses of 50 and 100 mg/kg/day, and the ascaridole, one of its main constituents, inhibited the tumor development by 33.9% and 33.3%, at doses of 10 and 20 mg/kg/day, respectively [6]. Herein, the EO from the leaves of C. matourensis inhibited the tumor mass by 34.6% to 55.9% at doses of 40 and 80 mg/kg/day, respectively, and although the EO was less potent than the positive control 5-fluorouracil, the EO presented similar or superior efficacy to that observed for it. The positive control 5-fluorouracil inhibited the tumor mass by 44.2% at a dose of 10 mg/kg/day.
In conclusion, the EO from the leaves of C. matourensis has β-caryophyllene, thunbergol, cembrene, p-cymene, and β-elemene as major constituents. Moreover, the EO has an in vitro and in vivo anti-liver cancer effect. These data indicate the anticancer potential effect of this plant.

Essential Oil Extraction
After collection, the aerial parts were directly extracted by hydrodistillation in a Clevenger-type apparatus. For this, 300 g of fresh and crushed material was extracted for a period of 4 h in 1000 mL of ultrapure water (18.2 MΩ). Then, the obtained oils were extracted with CH 2 Cl 2 , dried over anhydrous Na 2 SO 4 and filtered through a nylon membrane (pore size 0.22 µm, Whatman, Maidstone, UK). The resulting EO were weighed in vials and stored at −4 • C prior to chemical analysis.

Chemical Analysis
The qualitative analysis was performed by gas chromatography coupled to mass spectrometry (GC-MS) with an equipment model GCMS/QP2010 Plus (Shimadzu, Kyoto, Japan) using a selective detector and a capillary column Rtx-5 MS (30 m × 0.25 mm × 0.25, Restek). Helium was used as carrier gas with a flow of 1.02 mL/min. Injections (1 µL) were performed with EO solutions at 2 mg/mL in CH 2 Cl 2 using a split ratio of 1:50. The column temperature program was 60 to 280 • C with gradual increase of 3 • C/min. The temperatures of the injector and the ion source were 220 • C and 260 • C, respectively. Preliminary identifications of the constituents were performed based on comparison of experimental spectra with those stored in the Wiley 8th edition library (similarities > 90%). The confirmation of the identifications was performed by the calculation of the retention indexes (RI) according to the Van den Dool and Kratz equation [33] in comparison to a homologous series of linear hydrocarbons (C7-C30). A semi-quantitative analysis was performed to obtain the relative amount of each component of the EO. For this procedure, a gas chromatography with flame ionization detection (GC-FID) system, model GC2010 (Shimadzu) equipped with a Rtx-5 capillary column, was used. The same conditions of the GC-MS analysis were employed to ensure reproducibility. Relative amounts (%) were calculated in relation to the total area of the chromatogram.

Cells
Human cancer cell lines MCF-7 (breast adenocarcinoma), HCT116 (colon carcinoma), HepG2 (hepatocellular carcinoma), HL-60 (promyelocytic leukemia), and human non-cancer cell line MRC-5 (lung fibroblast) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured as recommended by the ATCC. Cell viability was examined via trypan blue exclusion method for all experiments.

Cell Viability Assay
Cell viability was measured by the Alamar blue assay and was performed as previously described [34][35][36]. For all experiments, cells were plated in 96-well plates. The EO was dissolved in dimethyl sulfoxide (DMSO, Vetec Química Fina Ltd., Duque de Caxias, RJ, Brazil) and added to each well and incubated for 72 h. Doxorubicin (purity ≥ 95%, doxorubicin hydrochloride, Laboratory IMA S.A.I.C., Buenos Aires, Argentina) was used as the positive control. At the end of the treatment, 20 µL of a stock solution (0.312 mg/mL) of resazurin (Sigma-Aldrich Co., Saint Louis, MO, USA) was added to each well. Absorbances at 570 and 600 nm were measured using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Annexin-V/PI Staining Assay
For apoptosis quantification, FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used, and the analysis performed according to the manufacturer's instructions. Cell fluorescence was measured by flow cytometry with a BD LSRFortessa cytometer. For flow cytometry analyses, 10 4 events were recorded per sample with the BD FACSDiva Software (BD Biosciences) and Flowjo Software 10 (Flowjo LLC, Ashland, OR, USA). Cellular debris was omitted from the analysis.

Internucleosomal DNA Fragmentation and Cell Cycle Distribution
The internucleosomal DNA fragmentation and cell cycle distributions were determined using propidium iodide (PI) (Sigma-Aldrich Co. St. Louis, MO, USA) in permeabilized cells, and performed as previously described [37]. Cell fluorescences were measured by flow cytometry as described above.

Animals
A total of 50 C.B-17 severe combined immunodeficient (SCID) mice (females, 25-30 g) were obtained and maintained at Gonçalo Moniz Institute-FIOCRUZ animal facilities (Salvador, Bahia, Brazil). Animals were housed in cages with free access to food and water. All animals were subjected to a 12:12 h light-dark cycle (lights on at 6:00 a.m.). A local animal ethics committee approved the experimental protocol employed (number #06/2015).

Human Hepatocellular Carcinoma Xenograft Model
HepG2 cells (10 7 cells per 500 µL) were implanted subcutaneously into the left front armpits of the mice. At the beginning of the experiment, mice were randomly divided into four groups. Group 1: animals received injections of vehicle with 5% DMSO solution (n = 20). Group 2: animals received injections of 5-fluorouracil (10 mg/kg/day, Sigma-Aldrich, n = 10). Group 3: animals received injections of the EO at 40 mg/kg/day (n = 10). Group 4: animals received injections of the EO at 80 mg/kg/day (n = 10). These dosages were selected based on previous works using EO in tumor models in mice [25,38]. Beginning 1 day after tumor implantation, the animals were treated through the intraperitoneal route for 21 consecutive days. One day after the end of the treatment, animals were anesthetized, and peripheral blood samples were collected from the brachial artery. Animals were euthanized by anesthetic overdose, and tumors were excised and weighed.

Toxicological Evaluation
The mice were weighed at the beginning and end of the experiment to evaluate the toxicological effects. Animals were observed for signs of abnormality throughout the study. A hematological analysis was performed using the Advia 60 hematology system (Bayer, Leverkusen, Germany). Livers, kidneys, lungs, and hearts were removed, weighed, and examined for signs of gross lesion formation, color change, and/or hemorrhaging.

Statistical Analysis
Data were presented as means ± standard error of the mean (SEM) or as IC 50 values with 95% confidence intervals obtained by nonlinear regressions. Differences among the experimental groups were compared through analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P < 0.05). All statistical analyses were performed using GraphPad Prism (Intuitive Software for Science; San Diego, CA, USA).