Evaluation of cytotoxic and antitumor activity of perillaldehyde 1 , 2-epoxide

1 Department of Medicine, Federal University of Sergipe, 330 Governador Marcelo Déda Ave, 49400-000 Lagarto, SE, Brazil. 2 Laboratory of Nanotechnology and Nanomedicine, Tiradentes University, 300 Murilo Dantas Ave, 49032-490 Aracaju, SE, Brazil. 3 Department of Physiology, Federal University of Sergipe, Marechal Rondon Ave, 49100-000 São Cristóvão, SE, Brazil. 4 Laboratory of Morphology and Experimental Pathology, Tiradentes University, 300 Murilo Dantas Ave, 49032-490 Aracaju, SE, Brazil. 5 Department of Physiology and Pharmacology, Federal University of Ceará, Brazil. 6 Department of Pharmacy, Federal University of Sergipe, Marechal Rondon Ave, 49100-000 São Cristóvão, SE, Brazil. 7 Department of Pharmaceutical Sciences, Federal University of Paraíba, Campus I Lot. Cidade Universitaria, 58033455 João Pessoa, PB, Brazil.


INTRODUCTION
Cancer is the second most common cause of death in the world, surpassed only by cardiovascular disease and is also known as malignant tumor characterized by abnormal growth and proliferation of cells (Amaral et al., 2015;Polu et al., 2015).It is a frightful disease because the patient suffers pain, disfigurement, and loss of many physiological processes.Cancer may be uncontrollable and incurable and may occur at any time at any age in any part of the body (Umadevi et al., 2013).Natural products, especially constituents of essential oils from medicinal plants have been successful in treating various disorders in traditional medicine (Chinta et al., 2015;Sousa, 2015).Compounds of natural origin have provided new and potential leads to cancer chemotherapy, and many of them are the drug of choice in cancer treatment (Polu et al., 2015).Natural source products have offered many useful anticancer agents in current use, such as the plant-derived drugs vinblastine, irinotecan, topotecan, etoposide, and paclitaxel (Bhanot et al., 2011;Qurishi et al., 2011).
Sources are still available in abundance and offer the best possibilities of finding substances of therapeutic interest (Butler, 2008).Although there are several cancer therapies, an ideal anticancer drug has not been discovered, and numerous side effects limit treatment.However, research into new drugs has revealed a variety of new chemical structures and potent biological activities that are of interest in the context of cancer treatment (Carvalho et al., 2015).
In fact, several compounds derived from natural products are in phase clinical trials mainly for cancer treatment, such as the monoterpene S-(-)-perillyl alcohol which shows cytotoxic and antitumor activity in various experimental models (Andrade et al., 2015;Andrade et al., 2016).Perillyl alcohol, a naturally occurring monoterpene found in essential oils of peppermint and lavender has been widely researched it is useful agents against a variety of human tumor cell lines (Andrade et al., 2015;Chen et al., 2015;Garcia et al., 2015).
In this context, the development of new derived compounds from natural plants and their analogues for anticancer and antitumor activities, and for this, the significant challenge is to synthesize, isolate and characterize novel derivatives based on bioactivity and mechanisms of action of these compounds.However, there is a continuing need for research and development of new anticancer drugs, drug combinations and Andrade et al. 591 chemotherapy strategies, by methodical and scientific exploration of an enormous pool of synthetic, biological and natural products (Umadevi et al., 2013).Therefore, considering the perillyl alcohol anticancer bioactivity, this study aimed to evaluate its chemical analogue, perillaldehyde 1,2-epoxide, on antitumor activity, toxicology effects and cytotoxicity mechanism (Figure 1).

Cell lines and MTT assay
For cytotoxicity assays, four human cancer cell lines HCT-116(colon carcinoma), OVCAR-8 (ovarian adenocarcinoma), SF-295 (glioblastoma), and HL-60 (Promyelocytic leukemia) were acquired from National Cancer Institute, Bethesda, MD, USA and evaluated for monoterpene perillaldehyde 1,2-epoxide.Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin, and incubated at 37°C in a 5% CO2 atmosphere.The MTT method, cytotoxic described by Mossman (1983), was used to evaluate the activity of the monoterpene perillaldehyde 1,2-epoxide against four human cancer cell lines.For the experiments, the cells were *Corresponding author.E-mail: luciana.nalone@hotmail.com.placed in 96-well plates (0.1 × 10 6 cells/ml in 100 μl medium).After 3 days(72 h) of incubation, the perillaldehyde 1,2-epoxide dissolved in dimethyl sulfoxide (DMSO 0.7%), at a final concentration of 10.60 μM, was added to each well and incubated for 3 days (72 h) at 37°C in a 5% CO2 atmosphere (three independent experiments, performed in triplicate).DMSO at 1% was used as negative control and doxorubicin at 0.55 μM was used as positive control (purity > 98%; Sigma Chemical Co., St. Louis, MO, USA).At the end of incubation, the plates were centrifuged at 5000 rpm for 10 min, and the supernatants were removed.A 150 μl of an aqueous MTT solution containing 0.5 mg/ml MTT was added to each well and incubated for three hours at 37°C in a 5% CO2 atmosphere.Cell viability was measured by the ability of cells valves to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO, USA) to a purple formazan product.
After incubation, the precipitate was dissolved in 150 μl DMSO, and absorbance 595nm was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc.).The absorbance values of these tests will be expressed as cell growth inhibition percentage (GI%) by the following formula: [GI% = 100 − [(T/NC) × 100%].NC is the Absorbance of the negative control, and T is the Absorbance of test compound (perillaldehyde 1,2-epoxide).
The median inhibitory concentration able to induce 50% of maximal effect (IC50) of the perillaldehyde 1,2-epoxide was determined after evaluation of GI%.All cells were retested using the same protocol above with a varying concentration of compound (0 to 150.60 µM) (dos Santos Júnior et al., 2010;Ribeiro et al., 2012).

Trypan blue dye exclusion assay
The human cancer cell line HL-60 was used at a concentration of 0.3 x 10 6 cells/ml, incubated for 3 days (72 h) with perillaldehyde 1,2-epoxide (4.85, 9.70 and 19.40 µM) and examined on an inverted microscope.At the end of incubation 90 µl was withdrawn from the cell suspension and added to 10 µl of trypan blue.The cells were differentiated into viable and non-viable and counted in a Neubauer chamber.A Doxorrubicin 0.55 µM was used as positive control (Veras et al., 2004).

Morphological analyses using a fluorescence microscope
The human cancer cell line HL-60 was used at a 0.3 x 10 6 cells/ml, incubated for 1 day (72 h) with perillaldehyde 1,2-epoxide (4.85, 9.70 and 19.40 µM).The cell suspension was transferred to an eppendorf tube and centrifuged for 5 min at low speed (145 × g).The supernatant was discarded, and the cells were resuspended in 20 µl of PBS solution.Then 1 µl of aqueous acridine orange/ethidium bromide solution (AO/EB, 100 μg/ml) was added to each tube, and an aliquot of these cells transferred to a slide and mounted with a coverslip and then brought to a fluorescence microscope for observation of cellular events (apoptosis and necrosis).Doxorubicin (0.55 µM) was used as positive control (Geng et al., 2005).

Morphological analysis using an optical microscope
The HL-60 cell line, plated at a concentration of 0.3 x 10 6 cells/ml, was incubated for 72 h with the compound (4.85, 9.70 and 19.40 µM) and examined under a microscope inverted.After that, 50 μl of cell suspension was added to the slide of the centrifuge (Cytospin™), to observe the morphology of the treated cells.After cell adhesion to the blade, fixing was done with methanol for 1 min and was first used hematoxylin staining, followed by eosin.The morphological changes were observed under optical microscope.Doxorubicin (0.55 µM) was used as positive control (Veras et al., 2004).

Hemolytic assay
To evaluate hemolytic activity, blood was collected from three mice of Swiss orbital path (anesthetized with isoflurane 1.5%) and diluted 1:30 in saline (0.85% NaCl + 10 mM CaCl2).The erythrocytes were washed two times in saline by centrifugation (15 g / 3 min.),resuspended in saline to obtain a suspension of erythrocytes 2% and the assay was performed in a 96-well plate (Jimenez et al., 2003).The single concentration of compound (500 µg/ml) was added to the suspension of red blood cells (Bezerra et al., 2005;Kang et al., 2009;Pita et al., 2012).Mixtures were incubated on a mixer for 60 min and then centrifuged at 875 × g for 5 min.Triton X-100 (1%) was used as the positive control.The absorbance of the supernatants was determined at 540 nm using spectrophotometrically.Triton X-100 (1%) was used as the positive control.

In vivo antitumor activity assay
To evaluate the in vivo antitumor activity, 60 male mice, weighing 26-31 g were used; they were purchased from bioterium of the Federal University of Sergipe, Brazil.The animals were maintained under laboratory conditions of temperature, humidity, and light with food and water ad libitum.The experimental protocol was submitted and approved by the Animal Care and Use Committee at the Federal University of Sergipe (CEPA: 16/2014).Ten-day-old sarcoma 180 ascites tumor cells (2 x 10 6 cell / 500 μl) were implanted subcutaneously into the left axillary region of the experimental mice (Bezerra et al., 2006).The animals were divided into 6 groups of 10 animals in polypropylene cages.One day after inoculation, was administered intraperitoneally in group one 5% DMSO (negative control-NC), groups two, three, four and five perillaldehyde 1,2-epoxide as doses de 25, 50, 100, 200 mg/kg/day respectively dissolved in 5% DMSO (test compounds) and group six 5-fluorouracil (5-FU, purity > 99%; Sigma Chemical Co.) 25 mg/kg/day (positive control).72 h after the last day of treatment under isoflurane inhalation (1.5%, calibrated vaporizer) anaesthesia, peripheral blood samples were collected from the retro-orbital plexus (toxicological analyses), the animals were euthanized by cervical dislocation, and the tumors, livers, spleens, and kidneys were excised and weighed.Inhibition of tumor growth (%) was expressed by the following equation (Bezerra et al., 2006): NC = mean tumor weight of the negative control group TC = mean tumor weight of the test compound treated group

Systemic toxicological analyses
Control groups (group one and six) and the groups that showed antitumor activity in vivo against sarcoma 180 were submitted to an evaluation by six toxicological parameters: variation in body mass, organ weights, liver, renal and hematologic parameters and histopathological analyses.First, the mice were weighed at the beginning and end of the experiment, and the animals were observed for signs of abnormalities throughout the study.Second, the livers, kidneys, and spleens were removed and weighed Andrade et al. 593 after euthanasia.Third and fourth parameters using obtained plasma peripheral blood samples of the mice and Clinical Chemistry® kits (Abbott; Architect C 8000) were evaluated liver function measured by aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and renal function measured by urea and creatinine.The fifth parameter was hematological analysis, an aliquot of blood from each animal was placed in EDTA, and total leucocyte counts were determined by standard manual procedures using optical microscopy.The last parameter was performed after 10% formaldehyde fixation, the spleens, liver, and kidneys were dehydrated in alcohol, diaphanized in xylene and paraffin-embedded.Subsequently, 5-μm-thick histological sections were obtained and stained with hematoxylin and eosin.Histological analyses were performed under optical microscopy (Amaral et al., 2016;Dória et al., 2016).The mice were weighed at the beginning and end of the experiment, and they were observed for signs of abnormalities throughout the study.At the end of the investigation, the animals were anesthetized with ether anesthesia, and peripheral blood samples by retro-orbital plexus were collected.
For hematological analysis, an aliquot of blood from each animal was placed in EDTA, and total leucocyte counts were determined by standard manual procedures using optical microscopy.Serum samples were obtained to evaluate liver function (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)), and renal function (urea and creatinine) using Clinical Chemistry® kits (Abbott; Architect C 8000).Then, the animals were sacrificed in a CO2 chamber, and the spleens, liver, and kidneys were weighted and fixed with 10% formaldehyde.The organs were dehydrated in alcohol, diaphanized in xylene and paraffin-embedded.Subsequently, 5-μm-thick histological sections were obtained and stained with hematoxylin and eosin.Histological analyses were performed under optical microscopy (Amaral et al., 2016;Dória et al., 2016).

Statistical analysis
The results were expressed as the mean ± SEM, and the differences between the experimental groups were analyzed using ANOVA of unidirectional analysis of variance followed by the Student Newman-Keuls test.Significant values of p < 0.05 were considered.All statistical analyses were performed using GraphPad program® (Intuitive Software for Science, San Diego, CA, USA).

MTT assay
Compounds of natural origin have provided new and potential leads to cancer chemotherapy, and many of them are the drug of choice in cancer treatment (Prakash et al., 2013).Natural products are important sources of chemical structures, and these will be used as templates for construction of new compounds with improved biological properties (Mann, 2002).Following this trend, the perillaldehyde 1,2-epoxide from the evaluation of the cytotoxicity of structurally correlated p-menthane (%) = [(NC -TC) / NC] x 100 derivatives described by Andrade and collaborators was developed (Andrade et al., 2015).
Based on a similar study performed by Andrade et al. (2015), with 18 derivative compounds of perillyl alcohol, compound perillaldehyde 8,9-epoxide was the pmenthane derivative with the highest cytotoxic activity for the human cancer cell lines HCT-116, OVCAR-8, and SF-295.Comparing the perillaldehyde 1,2-epoxide with the perillaldehyde 8,9-epoxide, position isomers with high cytotoxicity in vitro, both substances have a skeleton pmenthane containing an aldehyde group and an epoxide group in its chemical structure.The replacement of the hydroxyl group with the aldehyde group and adding the epoxide group the structure of perillyl alcohol (GI = 95.82,91.68, 90, and 92%, respectively) resulted in an increasein GI% perillaldehyde 1,2-epoxide (GI = 99.46,99.37 and 95.66%, respectively) and perillaldehyde 8,9epoxide (GI = 98.64, 96.32 and 99.89%, respectively).This result suggests that the presence of these two functional groups in the compounds may be contributing to this higher biological effect.
Thus, the compound in this study was promoted for the determination of median inhibitory concentration able to produce 50% of maximal effect (CI 50 ) to verify the potency of the compound.For this, were used the same cell lines and measurement methods used to determine the GI%, varying only the concentration of compound between 0 to 150.60 μM.The perillaldehyde 1,2-epoxide exhibits values IC 50 in the range of 9.70 to 21.99 μM in the HCT-116 and SF-295 cell lines, respectively.Doxorubicin, used as the positive control, showed IC 50 values ranging from 0.02 to 1.95 μM for HCT-116 and OVCAR-8 cell lines, respectively (Table 1).For this test, considered as a promising candidate for antineoplastic activity, substances that present lower IC 50 values or equal to 24.10 μM (Suffness and Pezzuto, 1990).Therefore, the compound perillaldehyde 1,2-epoxide is a potential candidate for anticancer activity and eligible for progression of studies.

Trypan blue dye exclusion assay
The perillaldehyde 1,2-epoxide showed higher cytotoxicity front leukemic cell line HL-60 (GI=99.71%±2.43).Faced with this result, three concentrations of perillaldehyde 1,2-epoxide, ½ IC 50 (4.85μM), IC 50 (9.70μM) and 2 × IC 50 (19.40μM) were chosen against HL-60 for evaluation of antiproliferative effects.The analysis of cell viability in HL-60 leukemic cell lines was performed by trypan blue exclusion after 72 h of exposure.Trypan blue exclusion test allows separately quantify the viable cells of cells killed by the test compound (Barros et al., 2013).Perilladehyde 1,2epoxide caused a significant reduction in the number of viable cells (p < 0.05) and increase in the number of nonviable cells (p < 0.05) at the concentrations of 4.85, 9.70 and 19.40 µM (Figure 2).Studies have shown that compounds can interact with MTT, inhibiting the reduction of MTT and may produce a false positive result.Therefore, the use of other viability assays, such as trypan blue exclusion is indicated before progression of the studies (Trevisi et al., 2006;Pita et al., 2012).Then, we confirm the cytotoxic activity of perilladehyde 1,2epoxide front HL-60 by two methods (MTT and Trypan blue exclusion).

Morphological analyses using a fluorescence and light microscopy
For the identification of the cell death process induced by perilladehyde 1,2-epoxide against HL-60 two assays, acridine orange/ethidium bromide was used and analysed by fluorescence microscopy and optical microscopy using hematoxylin-eosin coloration.After 72 h of incubation of the cells with compound at concentrations of 4.85, 9.70 and 19.40 µM and acridine orange/ethidium bromide stained, was observed as a result of a reduction in the number of viable cells and increase of cell death by apoptosis statistically significant (p < 0.05).This effect was observed when compared to the negative control group at all three concentrations of the compound tested (Figure 3).
In the morphological analysis by hematoxylin-eosin coloration of treated cells with perilladehyde 1,2-epoxide, in all concentrations tested there were signs of death by apoptosis as was also observed in the test with acridine orange/ethidium bromide (Figure 4).Other compounds structurally similar to the perilladehyde 1,2-epoxide as carvone (Aydın et al., 2015), carvacrol (Jaafari et al., 2009), limoneno (Sahin et al., 1999), perillyl alcohol (Clark, 2006) and perillic acid (Yeruva et al., 2007) also induce cell death by apoptosis in tumor cell lines.Cell death processes have well defined morphologic characteristics.Apoptosis includes cells with pyknotic appearance, condensation of chromatin, the fragmentation of the nucleus and the shedding of apoptotic bodies, vacuoles containing cytoplasm and intact organelles (Nikoletopoulou et al., 2013).Apoptosis is the cell death mechanism better known, used as an essential target for cancer therapy, by anticancer drugs such as the colcichine, taxanes and vinca alkaloids (Su et al., 2013;Topham and Taylor, 2013).(HL-60), using acridine orange/ethidium bromide after one day (72 h) of incubation.Viable cells were identified with a black bar, apoptotic cells were detected with gray bar, and necrosis cells were exposed with a white bar.The negative control (NC) was treated with 0.1% DMSO used for diluting the test compound.Doxorubicin, 0.55 µM was used as the positive control (PC).Data are presented as the mean values ± standard error of the mean of three independent experiments performed in duplicate.
When concentration of 19.40 µM of perilladehyde 1,2epoxide was used, concomitantly to cell death by apoptosis, was observed cell death by necrosis in tests performed with acridine orange/ethidium bromide and hematoxylin-eosin (Figures 3 and 4).Several treatments for malignant neoplasms can induce cell death by activation of necrosis to include photodynamic treatment (PDT), by the alkylation of agents harmful to DNA, and several other chemical compounds or substances such as apoptolidine, β-lapachone and honokil also appear to promote the death of cancer cells by necrosis (Zong and Thompson, 2006).Morphologically, the necrotic cells are characterized by the swelling of organelles, rupture of the plasma membrane (cell lysis), the nucleus becomes distended (nucleus intact) and usually followed by inflammatory reactions (Nikoletopoulou et al., 2013).Necrosis appears to be a limiting factor for increasing the concentration of the compound, but additional testing with higher levels has to be performed for more conclusions that are accurate.
Thus, the compound perilladehyde 1,2-epoxide can be considered a promising candidate for evaluation in vivo antitumor activity, by present cytotoxic activity predominant cell death by apoptosis, being subject to necrotic cell death at the highest concentration.

Evaluation of hemolytic activity
The evaluation of the hemolytic effect of perillaldehyde 1,2-epoxide was performed using erythrocytes Swiss mice, according to the methodology described by Costa-Lotufo and collaborators (Costa-Lotufo et al., 2002).This technique allows evaluating the potential of test compound in causing damage to the plasma membrane of the cell, either by the formation of pores or by the full rupture of it.However, this assay, we observed the absence of hemolytic activity in the tested concentration 500 μg/ml (Data not presented).These results suggest a possible selectivity for perillaldehyde 1,2-epoxide and enabling progression of studies related to the anticancer activity.

Tumor Sarcoma 180
Being the perillaldehyde 1, 2-epoxide cytotoxic against four human cancer cell lines (IC 50 < 4,0 μg/ml) and having a predominant cell death of apoptosis, we decided to investigate the possible in vivo antitumor activity of the test compound.Based on the use of experimental tumors for the identification of new products with potential anticancer, the antitumor activity of perillaldehyde 8,9epoxide was performed using sarcoma 180 tumors (original Swiss mice tumor, transplantable, and wellcharacterized experimental model) and treated by intraperitoneal route once a day for seven consecutive days (Britto et al., 2012;Yang et al., 2016).
The activity of perillaldehyde 1,2-epoxide on animals transplanted with sarcoma 180 tumor cells is presented in  When comparing the PC group to the groups treated with test compound, statistical alterations were observed for the groups 25, 50 and 100 mg/kg/day (p < 0.05) and no statistically significant changes for the group 200 mg/kg/day (p > 0.05).These results indicate that dose of 100 and 200 mg/kg/day the perillaldehyde 1,2-epoxide has antitumor activity in vivo against the sarcoma 180 with more significant action at the highest dose.
Other monoterpenes p-menthane as perillaldehyde 8,9epoxide and perillyl alcohol were also submitted to evaluation of antitumor activity in vivo using the same experimental tumor model.Both compounds were tested at doses of 100 and 200 mg/kg/day, demonstrating tumor mass growth inhibition rates of 38.4 and 58.7% for the perillaldehyde 8,9-epoxide and 35.3 and 45.4% for the perillyl alcohol, respectively (Andrade et al., 2016).Compared to the data obtained in this study with those obtained by Andrade and collaborators (Andrade et al., 2016) it was observed proximity between tumor mass growth inhibition rates and therefore a similar antitumor in vivo potency.The alcohol perillyl has been studied quite a long time to be able to inhibit the growth of tumor cells in cell culture and exert cancer preventive and therapeutic activity in a variety of animal tumor models.
Furthermore, the perillyl alcohol has been successfully used intranasally in the treatment of patients with malignant brain tumors (Chen et al., 2015).Thus, the results so far found with perillaldehyde 1,2-epoxide are promising for progression of studies.

Toxicological analyses
It is known that most of the currently used anticancer drugs are cytotoxic to tumor cells, but they also have non-specific action, because they affect healthy cells, leading to an undesirable side effect (Sun and Peng, 2008).Therefore we decided to evaluate toxicological characteristics of mice with tumor sarcoma 180 undergoing treatment with perillaldehyde 1,2-epoxide at doses of 100 and 200 mg/kg/day, to assess the cost and benefits of intervention.For this, some toxicological parameters were evaluated as variation in body mass, organ weights (liver, spleen, and kidney), liver (AST and ALT), renal (urea and creatinine), hematologic (total leukocytes) parameters and histopathological analyses (Dória et al., 2016).
There was no significant change (p > 0.05) in organ weights (liver, spleen and kidney), liver (AST and ALT) and renal (urea and creatinine) parameters in the groups treated with perillaldehyde 1,2-epoxide at doses of 100 and 200 mg/kg/day when compared with NC group (Table 2).These data are significant since many anticancer drugs cause changes in renal function, liver function and spleen organ volume as a result of the lack of specificity of antineoplastic, inducing severe adverse effects (Bezerra et al., 2008;Amaral et al., 2016).
When analyzing the variation in body mass and total leukocyte count, a significant reduction in body mass (p < 0.05) and a decrease in total leukocytes were observed in the groups treated with perillaldehyde 1,2-epoxide at one dose of 200 mg/kg/day compared to NC group.The group administered with compost test at a dose of 100 mg/kg/day no significant change (p > 0.05) the mass variation but significant decrease in total leukocytes (p < 0.05).These data can be considered a limiting factor for the administration of higher doses of test compound but does not prevent the advancement of studies because adverse effects such as body weight loss and leukopenia are common in some marketed antineoplastics (Cao et al., 1998;Zamagni et al., 1998).An example of these adverse effects is observed in the use of 5-FU 25 mg/kg/day as a positive control in the present paper, where a significant reduction (p < 0.05) in the variation of body weight, spleen weight and some leukocytes was checked.
Histopathology is a test used to aid in data biochemical and microscopic analysis, detecting any morphological change or characteristic lesion in the evaluated tissues.Histopathological examination was performed on organs, liver, spleen, and kidney of animals with sarcoma 180 and all tissue structures are substantially preserved in treated groups with perillaldehyde 1,2-epoxide at a dose of 100 and 200 mg/kg/day (Data not presented).

Conclusion
Therefore, the present study indicates that the perillaldehyde 1,2-epoxide exhibits high cytotoxic activity against the human cancer cell lines HCT-116, OVCAR-8, SF-295 and HL-60 with the possible predominant process of cell death by apoptosis and in vivo antitumor effects Figure 1.Chemical structure of perillaldehyde 1,2-epoxide.

Figure 2 .
Figure2.Effect of perilladehyde 1,2-epoxide on leukemic (HL-60) cell viability measured by the trypan blue dye exclusion after 72 h of incubation.The negative control (NC) was treated with 0.1% DMSO used for diluting the test compound.Doxorubicin, 0.55 µM was used as the positive control (PC).Data are presented as mean values ± S.E.M. of two independent experiments performed in duplicate.* p < 0.05 compared with the negative control by ANOVA followed by Student-Newman-Keuls test.

Figure 3 .
Figure3.Identification of cell death induced by perilladehyde 1,2-epoxide in cancer cell line (HL-60), using acridine orange/ethidium bromide after one day (72 h) of incubation.Viable cells were identified with a black bar, apoptotic cells were detected with gray bar, and necrosis cells were exposed with a white bar.The negative control (NC) was treated with 0.1% DMSO used for diluting the test compound.Doxorubicin, 0.55 µM was used as the positive control (PC).Data are presented as the mean values ± standard error of the mean of three independent experiments performed in duplicate.

Figure 4 .
Figure 4. Morphological analyses of the effect of perilladehyde 1,2-epoxide on HL-60 cancer cell line after 72 h of incubation, using optical microscopy.Figure A (negative control) was treated with the vehicle (0.1% DMSO), Figure B (positive control) was treated with doxorubicin (0.55 µM) and Figure C, D, and E were treated with perilladehyde 1,2epoxide in concentrations of 0.8, 1.6, 3.2 μg/ml respectively.Black arrows show a reduction of cell volume; the nucleus becomes distended, nuclear fragmentation and cellular debris.

Figure 5 .
Figure5.The mean tumor mass was 1.62 ± 0.05 g, 1.48 ± 0.07 g, 1.22 ± 0.16 g and 0.83 ± 0.15 g for animals treated with compost test at doses of 25, 50, 100, and 200 mg/kg/day, respectively.The NC group presented mean tumor mass of 1.76 ± 0.08 g.Statistically significant alterations (p < 0.05) were observed in mice treated with perillaldehyde 1,2-epoxide at concentrations of 100 and 200 mg/kg/day when compared with NC group, with tumor mass growth inhibition rates of 33.44 and 56.39%, respectively.The test compound at doses of 25 and 50 mg/kg/day showed no statistically significant alterations (p > 0.05) compared with NC group.The PC group presented tumor mass growth inhibition rates of 66.38% and mean tumor mass of 0.51 ± 0.06 g, statistically significant when compared with NC group.When comparing the PC group to the groups treated with test compound, statistical alterations were observed for the groups 25, 50 and 100 mg/kg/day (p < 0.05) and no statistically significant changes for the group 200 mg/kg/day (p > 0.05).These results indicate that dose of 100 and 200 mg/kg/day the perillaldehyde 1,2-epoxide has antitumor activity in vivo against the sarcoma 180 with more significant action at the highest dose.Other monoterpenes p-menthane as perillaldehyde 8,9epoxide and perillyl alcohol were also submitted to evaluation of antitumor activity in vivo using the same experimental tumor model.Both compounds were tested

Figure 5 .
Figure 5. Investigation of antitumor activity of perillaldehyde 1,2-epoxide (25, 50, 100, and 200 mg/kg/day, i.p.) in mice transplanted with experimental tumor sarcoma 180.Tumor mass (g) was identified with black bars and inhibition (%) of tumor growth with white bars.The negative control (NC) was treated with 5%, i.p. (diluiting the test compound).The positive control (PC) was administered with 5-Fluorouracil at 25 mg/kg/day, i.p.Data are presented as a mean ± standard error of the mean (n = 10 mice/group).*p < 0.05 compared with the NC group, and # p < 0.05 compared with the PC group by variance analysis, followed by the Student-Newman-Keuls test.

Table 1 .
Cell growth inhibition percentage (GI %) and values concentration able to inhibit 50% of cell growth (IC50) of monoterpene perillaldehyde 1,2-epoxide against four tumor cell lines.Data are presented as GI values in % and IC50 values in μM with their mean ± SEM.The data were collected in from three independent experiments, performed in triplicate and measured by the MTT assay after 72 hours of incubation.Doxorubicin was used as positive control.

Table 2 .
Toxicological parameters of the evaluation of the monoterpene perillaldehyde 1,2-epoxide.Data are presented as mean values ± SEM, n = 10 animals/group with tumor sarcoma 180.AST: aspartate aminotransferase; ALT: alanine aminotransferase.5% DMSO, i.p. was used as the negative control (NC).5-Fluorouracil at 25 mg/kg/day, i.p. was used as the positive control (NP).*p< 0.05 for all groups compared with the negative control using analysis of variance, followed by the Student-Newman-Keuls test.withoutpresenting substantial toxicity in experimental tumor model sarcoma 180.It is possible to continue the study for applicability of this compound in other tumor models or as prototypes for the development of antineoplastic agents.