Toxicity and antitumor potential of Mesosphaerum sidifolium (Lamiaceae) oil and fenchone, its major component

Aerial parts of Mesosphaerum sidifolium (L’Hérit.) Harley & J.F.B.Pastore (syn. Hyptis umbrosa) were collected in July 2015 in Maturéia, Paraíba state, Brazil. The species was identified by Dr. Maria de Fatima Agra. Voucher specimen number AGRA 6964 was deposited at the Herbarium Lauro Pires Xavier at the Federal University of Paraíba, Brazil.

The GC analysis was performed on a Shimadzu QP2000-PLUS-A gas chromatograph using fused silica capillary column DB-5 (30 mx 0.25 mm id, 0.25 mM film thickness). Helium was used as carrier gas at a flow rate of 1.0 mL/min. The oven temperature was programmed from 60° to 240° at 3 °C/min. The injector and detector temperatures were 220 °C and 230 °C, respectively.

Analysis by Gas Chromatography - Mass Spectrometry (GC-MS) was performed on a Shimadzu QP2000-PLUS system-Quadrupole MS, operating with ionization energy of 70 eV and fused silica capillary column DB-5 (30 mx 0.25 mm id, 0.25 mM film thickness) with helium as a carrier gas at a flow rate of 1 mL/min with a split. The temperatures of injector and detector were 220 °C and 230 °C, respectively. The column temperature was set from 60 °C to 240 °C at 3 °C/min. The substances identification was performed by comparing their mass spectra with the GC-MS database (62 Nist Research Library) and Kovats retention index [20]. Retention rates of the compounds were obtained by coinjection of the essential oil with a standard mixture of hydrocarbons (C9-C24), applying the equation of Van den Dool & Kratz [21].

Ehrlich carcinoma cell line was generously provided by Pharmacology and Toxicology Division, CPQBA, UNICAMP (Paulínia, SP, Brazil). The cells were maintained in the peritoneal cavities of Swiss mice in the Dr. Thomas George Bioterium (Research Institute in Drugs and Medicines/Federal University of Paraíba, Brazil).

The evaluation of acute preclinical toxicity for EOM was performed based on the “Guidelines for Testing of Chemicals” n° 423 from The Organisation for Economic Co-operation and Development [23]. Mice (n=3 females/group) were subjected to single doses of 300 or 2000 mg/kg of EOM intraperitoneally (i.p.), to the control group it was administered vehicle alone (5% (v/v) Tween 80 in saline). For toxicity detection, suggestive signs of Central Nervous System (CNS), or Autonomic Nervous System (ANS) activity were recorded. Careful observation was performed at the intervals: 0, 15, 30, and 60 min, after 4 h, and daily for 14 days. The dose responsible for the death of 50% of the experimental animals (LD₅₀) was estimated.

For the micronucleus assay, female mice (n=5/group) were treated (i.p.) with single dose of 150 or 300 mg/kg EOM [24]. A positive control group (cyclophosphamide at 50 mg/kg i.p.), and a negative control group (Tween 80 at 5% in saline), were included. After 48 h, the animals were anesthetized with sodium thiopental (40 mg/kg), and peripheral blood samples were obtained from the tail (10 μL), for the blood smears. For each animal, three blood smears were prepared, and a minimum of 2000 erythrocytes were counted to determine the number of micronucleated erythrocytes [25].

Five to seven-day-old Ehrlich tumor cells, 0.5 mL at 2.0 × 10⁶ cells/mL, were implanted in the peritoneal cavity of the female mice (n=6/group) [26]. One day after inoculation, EOM (50, 100 or 150 mg/kg) and its major component (30 or 60 mg/kg) were dissolved in 5% (v/v) Tween-80 in 0.9% (w/v) NaCl, and administered for nine consecutive days (i.p.). 5-FU (25 mg/kg) was used as a positive control. The healthy group (healthy mice) and tumor control group (mice bearing Ehrlich ascites carcinoma cells), were treated with 5% Tween-80 in 0.9% (w/v) NaCl. On the eleventh day, mice were kept fasting for 6 h, and peripheral blood samples from all groups were collected from the retro-orbital plexus under light sodium thiopental anesthesia (40 mg/kg), the animals were then euthanized.

The volume of ascitic fluid collected from the peritoneal cavity was expressed in milliliter (mL). It was determined the total viable cancer cells by trypan blue assay [26]. Tumor weights were measured by taking the mice’s weights before and after the collection of the ascitic fluid from peritoneal cavity, and expressed in grams (g).

For the determination of cell cycle phase distribution, 10⁶ cells from ascitic fluid of treated animals (groups: tumor control; 50, 100 and 150 mg/kg EOM; 30 and 60 mg/kg major component) were centrifuged at 230 g for 7 min. The supernatant was removed and the pellet was resuspended in 0.3 mL of hypotonic fluorocromic solution containing RNase (0,5 mg/mL), Triton-X (0,25%) and propidium iodide (PI) (0,25 mg/mL). Then, the analysis was performed by cytometric flow (BD FACSCalibur®, USA), a total of 10,000 events were obtained, and data were analyzed using WinMDI 2.9 software [27].

Body weights were registered at the beginning and end of the treatment while the water and food consumption was evaluated daily for the nine days of the treatment. Liver, spleen, thymus, and kidneys were weighed for the determination of their organ indices [organ weight (mg)/animal weight (g)]. For biochemical analysis, serum samples were used to determine the levels of urea, creatinine, and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). For the hematological analysis, heparinized whole blood was used to determine: hemoglobin (Hb) level, red blood cell (RBC) count, hematocrit (Hct), and the red cell indices mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) and, total and differential leukocyte counts. After weight determination and fixation in 10% (v/v) formaldehyde, portions of the livers and kidneys were cut into small pieces, then into sections of 3 μm, and stained with hematoxylin-eosin, and examined microscopically for lesions [28].

Data are presented as mean ± SEM. The differences between experimental groups were compared by analysis of variance (ANOVA), followed by Tukey’s test (p<0.05). The HC50 value and their 95% confidence intervals (CI 95%) were obtained by nonlinear regression. For the genotoxicity evaluation it was used the Mann-Whitney U test (p<0.05 was considered significant).

The essential oil was obtained by hydro distillation from the leaves of M. sidifolium with a yield of 0.6% compared to the fresh weight of its botanical material. The total percentage of volatile components identified was of 92.9%, comprising 57 components. The major compounds were fenchone (24.8%), cubebol (6.9%), limonene (5.4%), spathulenol (4.5%), β-caryophyllene (4.6%) and α-cadinol (4.7%) (Table 1).

EOM displayed a concentration-dependent haemolytic effect. The mean HC50 was 494.9 (494.2–495.6) μg/mL. Differently, fenchone did not induce hemolytic effect up to 3000 μg/mL (Fig. 1).

EOM 300 mg/kg did not induce death. Following the guideline N. 423 from OECD, we repeated the test using three additional animals, with the same dose. Again, no death was observed. At 300 mg/kg, EOM induced only weak toxicity signs, such as constipation and decreased urination, vocalization and hyperactivity, considered to be without clinical importance, since they disappeared after 60 min of administration. Then, we tested EOM 2000 mg/kg that induced severe symptoms and death of the three animals tested within the first minutes after treatment, therefore this dose was characterized as high toxicity (Table 2). The LD₅₀ was estimated approximately in 500 mg/kg.

EOM only in the highest dose (300 mg/kg) induced an increase in the number of micronucleated erythrocytes in peripheral blood (5.60 ± 0.40), as compared to the control group (2.80 ± 0.37). As expected, cyclophosphamide induced an increase of number of micronucleated erythrocytes (14.50 ± 2.60) (Table 3).

EOM (100 and 150 mg/kg) induced a significant decrease in tumor weight and volume, and tumor cell total count, when compared to the tumor control group. For fenchone (60 mg/kg), it was observed reduction in all analyzed parameters. Similar results were observed for the standard drug (5-FU, 25 mg/kg). Data are shown in Table 4.

In addition, there was an increase in survival time of all of groups, when compared with the tumor control group, however this effect was more pronounced at 100 mg/kg EOM (Fig. 2a) and 60 mg/kg fenchone (Fig. 2b).

EOM (100 and 150 mg/kg) exhibited significant changes in the distribution of Ehrlich carcinoma cells at different stages of the cell cycle (Fig. 3a). The results show that EOM 100 mg/kg induced increase in the S phase (58.27%), accompanied by the reduction in G2/M phase (9.1%). At the highest dose (150 mg/kg EOM), it was observed and increase in sub-G1 peak to 14.1% and in G0/G1 phase (65.6%), associated with descrease in S and G2/M phases (13.7% and 6.4%, respectively). For 5-FU, G0/G1 arrest was observed (84.3%), accompanied by the reduction in S (8.4%) and G2/M (4.8%) phases.

Fenchone also induced changes on cell cycle. There was an increase in S phase to 75.8%, associated with decrease in G2/M (3.5%) and G0/G1 (17.8%) phases (Fig. 3b).

EOM (50 mg/kg) produced an increase on water consumption, whereas at 150 mg/kg there was a decrease on feed consumption, when compared to respective healthy and tumor control groups. In addition, EOM (100 and 150 mg/kg) and 5-FU induced significant weight loss in the experimental animals after nine days of treatment. For fenchone (30 and 60 mg/kg), there was only an increase on water consumption (Table 5). In relation to organs index, there was an increase in liver index for tumor control group comparing with health animals; however, EOM reduced this parameter in relation to tumor control group. Fenchone did not restore the normal values for liver index. In addition, fenchone induced an increase in heart and thymus indexes (Table 6).

EOM induced no significant changes for AST and ALT activity levels. At 150 mg/kg there was only an increase in urea levels, without changes for creatinine levels. On the other hand, fenchone induced significant decrease in liver function parameters, AST and ALT (Table 7).

Regarding hematological evaluation, changes were observed in tumor control group, comparing with health animals, which included a decrease in red blood cells, hemoglobin and hematocrit. In contrast, EOM increased these parameters in relation to tumor control group, but these values remained as health animals. In addition, there was a leukemoid reaction in tumor control group; nevertheless, EOM (100 or 150 mg/kg) and 5-FU induced a significant reduction in this parameter. Yet, 5-FU induced lymphocyte increase and neutrophil count reduction compared to the tumor control group (Table 8). For fenchone, there was a re-established of the blood count parameters as health animals, comparing to the tumor control group. Similar to EOM, fenchone induced a significant reduction in total leukocytes number (Table 9).

No histopathological changes were observed in the kidneys of animals treated with EOM (Data not shown). The histopathological analysis of the livers showed a discrete, reactive lymphocytic, with rare and focal hepatocitolytic activity after treatment with EOM. It was also observed the presence of neutrocyte portite and capsulitis, besides hepatic tissue necrosis (Fig. 4a e b). In addition to these observations, also detected in the tumor control group, the liver of animals treated with EOM presented mild hyperplasia of Kuppfer cells (Fig. 4c). Animals treated with 5-FU experienced hepatic damage greater than those treated with the highest doses of EOM (Fig. 4d).