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Research Paper
Journal of Pharmacy And Pharmacology
Evaluation of the cytotoxic and antitumour effects of the essential oil from Mentha x villosa and its main compound, rotundifolone Ricardo G. Amarala, Cecília S. Fonsecab, Tayane Kayne M. Silvab, Luciana N. Andradea, Maria E. Françac, José M. Barbosa-Filhod, Damião P. de Sousad, Manoel O. Moraesc, Cláudia Ó Pessoac, Adriana A. Carvalhob and Sara Maria Thomazzia a
Department of Physiology, Federal University of Sergipe, São Cristóvão, bDepartment of Pharmacy, Federal University of Sergipe, Lagarto, Sergipe, Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará and dDepartment of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, Paraíba, Brazil c
Keywords antitumour; cytotoxic; essential oil; Mentha x villosa; terpenes Correspondence Sara Maria Thomazzi, Department of Physiology, Federal University of Sergipe, 49100-000, São Cristóvão (SE), Brazil. E-mail: [email protected] Received November 24, 2014 Accepted February 8, 2015 doi: 10.1111/jphp.12409
Abstract Objectives The aim of this study was to investigate the cytotoxic and antitumour effects of the essential oil from the leaves of Mentha x villosa (EOMV) and its main component (rotundifolone). Methods In-vitro cytotoxic activity of the EOMV and rotundifolone was determined on cultured tumour cells. In-vivo antitumour activity of the EOMV was assessed in sarcoma 180-bearing mice. Key findings The EOMV displayed cytotoxicity against human tumour cell lines, showing IC50 values in the range of 0.57–1.02 μg/ml in the HCT-116 and SF-295 cell lines, respectively. Rotundifolone showed weak cytotoxicity against HCT-116, SF-295 and OVCAR-8 cell lines (IC50 > 25.00 μg/ml). Tumour growth inhibition rates were 29.4–40.5% and 25.0–45.2% for the EOMV treatment by intraperitoneal (50–100 mg/kg/day) and oral (100–200 mg/kg/day) administration, respectively. The EOMV did not significantly affect body mass and macroscopy of the organs. Conclusions The EOMV possesses significant antitumour activity with low systemic toxicity, possibly due to the synergistic action of its minor constituents.
Introduction Cancer is a leading cause of death worldwide, accounting for 8.2 million deaths in 2012 with 14.1 million new cases.[1] An exceptionally difficult problem in cancer treatment is multidrug resistance, when cancer cells lose their sensitivity to multiple structurally different chemotherapeutics, leading to the search for alternative treatments, such as medicinal plants. Several studies have demonstrated anticancer activity for the essential oils obtained from medicinal plants.[2–4] Some essential oils of the genus Mentha, an important member of the family Lamiaceae, have been described in the literature for its cytotoxic properties, such as Mentha spicata L. and Mentha arvensis L.[5,6] The species Mentha x villosa Hudson, popularly known as ‘hortelã-da-folha-miúda’ or ‘hortelã-rasteira’, is an herb 1100
largely cultivated in northeastern Brazil, where it has widespread use as a condiment for meat and pasta, and as a salad ingredient.[7] This species also possesses ethnobotanical use in the treatment of worm, influenza, inflammation and pains in general, fever, intestinal infection, indigestion, sinusitis, ophthalmic problems, otalgia, menstrual colic, headache, migraine, and stroke.[8] The literature suggests that the essential oils obtained from Mentha x villosa have various biological activity, such as antinociceptive, hypotensive, spasmolytic, schistosomicidal, antimicrobial and larvicidal.[9–14] Rotundifolone (Figure 1), also known as piperitenone oxide, is a naturally occurring monoterpenic ketone of plant origin and an important chemical constituent of the essential oils of many Mentha species: Mentha rotundifolia
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1100–1106
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Antitumour activity of M. x villosa essential oil
O
O
Figure 1 Chemical structure of rotundifolone (1,2-epoxy-p-menth4(8)-en-3-one).
(L.) Huds., Mentha suaveolens Ehrh., Mentha longifolia (L.) Huds., M. spicata and M. x villosa.[15,16] Previous studies have demonstrated that this compound has similar activity to those found in the essential oil of M. x villosa as hypotensive, larvicidal, antinociceptive and schistosomicidal.[9,10,12,14,16] However, there are no studies about the cytotoxic and antitumour activity of the essential oil of M. x villosa and its main constituent. Based on the considerations above, we decided to investigate the effects of the essential oil of Mentha x villosa leaves (EOMV) and rotundifolone, assessing their cytotoxic or antitumour actions, as well as the systemic toxicity of the EOMV.
Materials and Methods
tial oil was submitted to preparative thin layer chromatography. The plates were developed three times using hexanes as eluent. When the plates were exposed to UV light (254 nm), rotundifolone was visualized as the major component of the essential oil. Rotundifolone was removed from the chromatographic plates and later recovered by means of extraction with dichloromethane, followed by filtration and evaporation to obtain a yellowish oil. The structural identification was made with infrared, 1H and, 13C nuclear magnetic resonance analysis, and in comparison with the literature data.[15]
Animals A total of 80 Swiss mice (females, 25–30 g), obtained from the central animal house of the Federal University of Sergipe, Brazil, was used. Animals were housed in cages with free access to food and water. All animals were kept under a 12:12 h light–dark cycle. Animals were treated according to the ethical principles for animal experimentation of the SBCAL (Brazilian Association of Laboratory Animal Science), Brazil. The Animal Studies Committee from the Federal University of Sergipe approved the experimental protocol (CEPA/UFS 24/2013).
Plant material
Cells
M. x villosa leaves were collected in the Medicinal Plant Garden of the Pharmaceutical Technology Laboratory of the Federal University of Paraíba, Brazil, in July 2008 (07°08′29″S, 34°50′48″W). The plant was authenticated and a voucher specimen has been deposited at the Herbarium Prisco Bezerra of the Federal University of Ceará, Brazil (Number 14996).
The cytotoxicity of the EOMV and rotundifolone was tested against OVACAR-8 (ovarian adenocarcinoma), HCT-116 (colon carcinoma) and SF-295 (glioblastoma) human cancer cell lines, all obtained from the National Cancer Institute, Bethesda, MD, USA. Cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, and were then incubated at 37°C in a 5% CO2 atmosphere. Sarcoma 180 tumour cells, which had been maintained in the peritoneal cavity of Swiss mice, were obtained from the Laboratory of Experimental Oncology at the Federal University of Ceará, Brazil.
Extraction of the essential oil Fresh leaves of Mentha x villosa (10 kg) were submitted to hydrodistillation for 8 h using a Clevenger-type apparatus, and an oil of yellowish colouration and characteristic odour of mint was obtained. Subsequently, the oil was dried over anhydrous sodium sulfate, filtered and stored at 4°C. The percentage of yield of the EOMV was 0.1%.[14] The same batch of the EOMV tested in this manuscript was analysed through gas chromatography-mass spectrometry (GC-MS), and the results were described by Lima et al.[14] The essential oil of Mentha x villosa leaves contained rotundifolone (70.96%), limonene (8.75%), germacrene D (3.81%), myrcene (3.10%), trans-caryophyllene (1.46%) and other constituents in a low amount.
Isolation of rotundifolone Rotundifolone was isolated from the EOMV using a procedure previously described by Almeida et al.[15] The essen-
In-vitro cytotoxic activity assay Tumour cell growth was determined by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product.[17] For all experiments, cells were seeded in 96-well plates (0.1 × 106 cells/ml in medium, 100 μl). After 24 h, the EOMV and rotundifolone obtained from the EOMV (0–25 μg/ml), dissolved in 0.07% DMSO, were added to each well (three independent experiments, performed in triplicate). Afterwards, the cells were incubated for 72 h at 37°C in a 5% CO2 atmosphere. Doxorubicin (0.003–0.25 μg/ml) was used as positive control. At the end
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of the incubation, the plates were centrifuged, and the medium was replaced by fresh medium (150 μl) containing 0.5 mg/ml MTT. Three hours later, the formazan product was dissolved in 150 μl DMSO, and absorbance was measured at 595 nm using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc., Fullerton, CA, USA).
In-vivo antitumour activity assay The in-vivo antitumour effect was evaluated using sarcoma 180 ascites tumour cells, following the protocol previously described, with minor modifications.[2] Ten-day-old sarcoma 180 ascites tumour cells (2 × 106 cells per 500 μl) were implanted subcutaneously into the right axillary region of mice. After 24 h, the essential oil was dissolved in 10% DMSO (vehicle) and administered intraperitoneally (50 or 100 mg/kg) or orally by gavage (100 or 200 mg/kg) once a day for seven consecutive days. At the beginning of the experiment, the mice were divided into groups (n = 10 animals/group): (1) intraperitoneal injection of vehicle; (2) intraperitoneal injection of 5-Fluorouracil (5-FU, 25 mg/kg/day); (3) intraperitoneal injection of the EOMV at 50 mg/kg/day; (4) intraperitoneal injection of the EOMV at 100 mg/kg/day; (5) the oral route (p.o., gavage) with vehicle; (6) gavage (p.o.) with cyclophosphamide (10 mg/ kg/day); (7) gavage (p.o.) with the EOMV at 100 mg/kg/ day; and (8) gavage (p.o.) with the EOMV at 200 mg/kg/ day. On day 8, under isoflurane inhalation (1.5%, calibrated vaporizer) anaesthesia, the animals were euthanized by cervical dislocation, and the tumours were removed and weighed. The inhibition ratio of tumour growth (%) was calculated by the following formula: inhibition ratio (%) = ((A – B) / A) × 100, where A is the average tumour weight of the vehicle group and B is the average tumour weight of the treated group.[2]
Systemic toxicological analysis Body mass loss, organ weight alterations and haematological analysis were determined at the end of the experiment above.[2] Body weights were determined at the
start and on the last day of treatment. Before euthanasia, 24 h after the last treatment day, peripheral blood samples of the mice were collected from the retro-orbital plexus under isoflurane inhalation (1.5%, calibrated vaporizer) anaesthesia. For haematological analysis, an aliquot of blood from each animal was placed in EDTA, and haematological parameters (total and differential leucocyte counts) were determined by standard manual procedures using light microscopy. After euthanasia, the livers, spleens and kidneys were dissected and weighed. The wet mass of each organ was expressed as grams per 100 g of body mass and compared with the vehicle group.
Statistical analysis Data are presented as mean ± standard error of mean or half maximal inhibitory concentration (IC50) values, and their 95% confidence intervals were obtained through nonlinear regression. The differences between experimental groups were compared using analysis of variance, followed by the Student–Newman–Keuls test (P < 0.05).
Results The cytotoxic activity of the EOMV and rotundifolone was evaluated against several human tumour cell lines: HCT116 (colon), OVCAR-8 (ovarian) and SF-295 (brain). The IC50 values are shown in Table 1. The EOMV displayed cytotoxicity against all tumour cell lines tested, showing IC50 values in the range of 0.57–1.02 μg/ml in the HCT116 and SF-295 cell lines, respectively. Rotundifolone showed IC50 values > 25.00 μg/ml in all tumour cell lines tested. Doxorubicin, used as the positive control, showed IC50 values ranging from 0.01 to 1.20 μg/ml for HCT-116 and OVCAR-8 cell lines, respectively. The next step of this study was the evaluation of the in-vivo antitumour activity of the EOMV. The effects of the EOMV on mice transplanted with sarcoma 180 tumour cells are presented in Figure 2. One day after the end of the treatment, the average tumour weight of the control (vehicle) mice was 1.90 ± 0.15 and 1.04 ± 0.07 g for intraperitoneal and oral administration, respectively (Figure 2).
Table 1 Cytotoxic activity of the essential oil from the leaves of Mentha x villosa (EOMV) and its main component (rotundifolone) on cancer cell lines Cell lines (IC50 values (μg/ml)) Treatment
HCT-116
OVCAR-8
SF-295
EOMV Rotundifolone Doxorubicin
0.57 (0.44–0.75) >25.00 0.01 (0.01–0.02)
0.86 (0.78–0.96) >25.00 1.20 (0.90–1.60)
1.02 (0.92–1.14) >25.00 0.24 (0.17–0.36)
Cell lines: HCT-116 (human colon carcinoma), OVCAR-8 (human ovarian adenocarcinoma) and SF-295 (human glioblastoma). Data are presented as IC50 values in μg/ml and their 95% confidence interval from three independent experiments, performed in triplicate, and measured by the MTT assay after 72 h of incubation.
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60
1.2 40
0.8
20
0.4
0
0.0 Vehicle
Cyclo
100
200
EOMV (mg/kg/day, p.o.) Figure 2 The effect of the essential oil from the leaves of Mentha x villosa on mice inoculated with sarcoma 180 tumour cells. The graphs show tumour mass (g) and tumour growth inhibition levels (%). The animals were treated by intraperitoneal (a) or oral (b) administration, starting 1 day after tumour implantation, for seven consecutive days. 5-Fluorouracil (5-FU, 25 mg/kg/day, i.p.) and cyclophosphamide (Cyclo, 10 mg/kg/day, p.o.) were used as the positive controls. Vehicle (10% DMSO) was used as the negative control. Data are presented as mean ± standard error of mean (n = 10 animals/group). *P < 0.05 compared with the vehicle group using analysis of variance, followed by the Student–Newman–Keuls test.
In the presence of the EOMV (50 or 100 mg/kg/day) by intraperitoneal administration, the average tumour weights were 1.34 ± 0.16 and 1.13 ± 0.15 g, respectively. In the presence of the EOMV (100 or 200 mg/kg/day) by oral administration, the average tumour weights were 0.78 ± 0.11 and 0.57 ± 0.11 g, respectively. Tumour growth inhibition rates were 29.4–40.5% and 25.0–45.2% for the EOMV treatment by intraperitoneal and oral administration, respectively. In both routes of administration, there were statistically significant differences between both doses in relation to the vehicle group (P < 0.05). 5-FU (25 mg/kg/day, i.p.) and cyclophosphamide (10 mg/kg/day, p.o.) reduced tumour mass by 69.4% and 59.5%, respectively. Systemic toxicological parameters were also examined in the EOMV-treated mice as shown in Tables 2 and 3. No sig© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1100–1106
1.25 ± 0.04 1.11 ± 0.06 1.30 ± 0.06 1.24 ± 0.04 0.52 ± 0.02 0.47 ± 0.03 0.60 ± 0.04 0.41 ± 0.04
1.22 ± 0.04 1.26 ± 0.06 1.18 ± 0.04 1.30 ± 0.03
Sarcoma 180 tumour cells (2.0 × 106cells/animal, s.c.) were implanted in mice. The animals were treated, starting 1 day after tumour implantation, for seven consecutive days. Data are presented as mean ± standard error of mean (n = 10 animals/group). *P < 0.05 compared with the vehicle group using analysis of variance, followed by the Student–Newman–Keuls test.
1.6
Cyclophosphamide
80
Inhibition (%)
Tumour mass (g)
2.0
100
5.06 ± 0.11 4.91 ± 0.31 5.65 ± 0.31 5.16 ± 0.16
Inhibition
0.9 ± 0.3 0.6 ± 0.3 0.3 ± 0.2 −0.4 ± 0.3
Tumour mass
– 100 200 10
(b) 2.4
5-Fluorouracil Oral route Vehicle (10% DMSO) EOMV
EOMV (mg/kg/day, i.p.)
0.58 ± 0.04 0.61 ± 0.03 0.56 ± 0.03 0.36 ± 0.03*
100
4.83 ± 0.25 4.72 ± 0.19 4.71 ± 0.15 4.99 ± 0.16
50
2.1 ± 0.4 0.9 ± 0.4 0.7 ± 0.6 −3.1 ± 0.5*
5-FU
50 100 25
0 Vehicle
–
0.0
Intraperitoneal route Vehicle (10% DMSO) EOMV
20
0.4
Spleen (g/100 g body mass)
40
0.8
Liver (g/100 g body mass)
60
1.2
The effect of the essential oil from the leaves of Mentha x villosa (EOMV) on body and organ weights of mice transplanted with sarcoma 180 tumour cells
1.6
Variation in body mass (g)
80
Inhibition (%)
Tumour mass (g)
2.0
100
Dose (mg/kg/day)
Inhibition
Treatment
Tumour mass
Table 2
(a) 2.4
Antitumour activity of M. x villosa essential oil
Kidney (g/100 g body mass)
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Antitumour activity of M. x villosa essential oil
Table 3
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The effect of the essential oil from the leaves of Mentha x villosa (EOMV) on the haematological parameters in peripheral blood
Treatment Intraperitoneal route Vehicle (10% DMSO) EOMV 5-Fluorouracil Oral route Vehicle (10% DMSO) EOMV Cyclophosphamide
Differential count of leucocytes (%)
Dose (mg/kg/day)
Total leucocytes (103 cells/μl)
Eosinophil
Lymphocyte
Neutrophil
– 50 100 25
8.8 ± 0.5 7.3 ± 0.5*# 6.2 ± 0.2*# 2.4 ± 0.4*
0.4 0.4 0.2 0.2
50.4 54.2 57.2 75.2*
44.2 39.6 37.0 21.2*
5.0 5.8 4.2 3.6
– 100 200 10
9.5 ± 1.1 8.3 ± 0.7 7.7 ± 2.6 6.3 ± 0.8
0.2 0.2 0.0 0.0
59.3 55.6 60.4 61.2
32.5 36.6 31.6 27.8
8.0 7.6 8.0 11.0
Monocyte
Sarcoma 180 tumour cells (2.0 × 106cells/animal, s.c.) were implanted in mice. The animals were treated, starting 1 day after tumour implantation, for seven consecutive days. Data are presented as mean ± standard error of mean and % (n = 10 animals/group). *P < 0.05 compared with the vehicle group and #P < 0.05 compared with the 5-fluorouracil group by analysis of variance, followed by Student–Newman–Keuls.
nificant changes were observed in body mass gain after administration of the EOMV by both routes (P > 0.05, Table 2). Also, no significant changes in the mass of livers, kidneys or spleens were seen in the essential oil-treated groups (P > 0.05). Cyclophosphamide also failed to significantly change the body mass and the mass of organs, compared with the vehicle group (10% DMSO). In contrast, 5-FU reduced the body mass and spleen organ mass (P < 0.05). Another systemic toxicological parameter examined was the blood leucocytes count, as shown in Table 3. In the peripheral blood from mice transplanted with sarcoma 180 tumour cells, the EOMV and 5-FU induced a decrease in total leucocytes (P < 0.05) after intraperitoneal administration. However, leucopenia was more pronounced in 5-FUtreated animals compared with the EOMV-treated groups (50 and 100 mg/kg/day, i.p.; P < 0.05). Different from the results observed after intraperitoneal administration, the EOMV and cyclophosphamide reduced tumour development (Figure 2) without inducing a disturbance in the leucocytes count when administered orally (Table 3).
Discussion In the present work, we demonstrate for the first time the in-vitro cytotoxic activity of the EOMV and its main component (rotundifolone) against tumour cell lines, and the in-vivo antitumour effect of the EOMV on mice transplanted with sarcoma 180 tumour cells as well as its systemic toxicity. Previous GC-MS performed by Lima et al. with the same batch of the essential oil used in this study showed the presence of rotundifolone (70.96%), limonene (8.75%), germacrene D (3.81%), myrcene (3.10%) and transcaryophyllene (1.46%) as major compounds.[14] Interest1104
ingly, the major component in the EOMV identified by other authors was also rotundifolone, despite variations in the percentage composition (58.74–95.87%).[12,18,19] Generally, the major components are found to reflect quite well the biophysical and biological features of the essential oils from which they were isolated, the amplitude of their effects being just dependent on their concentration when they were tested alone. However, it is possible that the activity of the main components is modulated by other minor components.[20,21] Therefore, besides the EOMV, we investigated the cytotoxic action of rotundifolone on cancer cell lines. According to the preclinical anticancer drug screening programme used in this study, an essential oil that shows IC50 values below 30 μg/ml and an isolated compound that shows IC50 values below 1 μg/ml are considered promising.[3,4] Therefore, we considered that the EOMV presents potent cytotoxic activity, and rotundifolone obtained from Mentha x villosa plant has weak cytotoxic activity against the tumour cell lines tested in the concentration range used. The potent cytotoxic activity of the EOMV demonstrated in this study might be attributed to the mixture of its other constituents. Many studies have demonstrated that several constituents found in the EOMV, including sabinene, β-pinene, myrcene, limonene and germacrene D show cytotoxic and anticancer activity justifying the in-vitro effect shown by the EOMV and its possible action in vivo.[20,22,23] Previous studies have demonstrated the synergistic action of the combination of natural compounds with anticancer activity. Rahman et al. demonstrated that the combination of two plant bioactive compounds, epigallocatechin gallate (a catechin) and [6]-gingerol (a major component of ginger), synergistically induces apoptosis and inhibits the proliferation of 1321N1 and LN18 cells (human glioblastoma cell lines).[24] The combination treatment of epigallocatechin gallate and curcumin (a phenolic
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Antitumour activity of M. x villosa essential oil
compound) showed synergistically cytotoxic action towards uterine leiomyosarcoma cells in vitro, and it reduced uterine leiomyosarcoma cells viability more than did the treatment with either drug alone.[25] Sarcoma 180 is an original mouse tumour, transplantable and well-characterized experimental model used in the research of in-vivo antitumour activity.[26] Currently, most of the drugs used in chemotherapy are administered by the parenteral route. However, there are few drugs that can be used by the oral route. Therefore, we decided to evaluate the antitumour activity of the EOMV using two different routes of administration: intraperitoneal (a parenteral route) and oral/gavage (an enteral route). The EOMV showed antitumour activity with both administration routes (i.p. and p.o.), but it was more potent by intraperitoneal administration. In fact, usually, the oral route is disadvantageous because of less absorption. Ingested drugs are subject to the first-pass effect, in which a significant amount of the agent is metabolized in the gut wall and the liver before it reaches systemic circulation. Thus, some drugs have low bioavailability when given orally.[2] Rotundifolone was not tested in an animal-bearing tumour due to its weak cytotoxic activity against the tumour cell lines. Most cancer chemotherapeutic agents are non-targeted in action and demolish rapidly dividing normal cells besides tumours, leading to extensive side effects. Some of the common clinical side effects include hepatic dysfunction, renal toxicity and haematopoietic depression. The liver plays a major role in metabolism and the kidney in the urinary system, and both have a number of functions in the body, including mostly detoxification and excretion of wastes, respectively. On the other hand, the spleen is an organ with important roles regarding the red blood cells and the immune system. Hepatic dysfunction induced by irinotecan, renal toxicity induced by docetaxel and haematopoietic suppression induced by 5-FU are such examples.[27–29] In addition, often, vomiting and diarrhoea are observed, leading to loss of body mass and malnutrition. Herein, variation in body mass, hepatotoxic and nephrotoxic effects, as well as changes in haematological parameters, were investigated.
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In this study, the treatment with the EOMV did not significantly affect the body mass or macroscopy of the organs (kidney, liver and spleen) by both routes, but induced a decrease in total leucocytes after intraperitoneal administration. Furthermore, we found that unlike the EOMV-treated groups, the treatment with 5-FU decreased the spleen mass, and it induced a leucopenia more pronounced with a disturbance in the differential count of circulating peripheral leucocytes. These results confirm the side effect of this immunosuppressive drug, clinically useful, which substantially increases the risk of infections.[30] This is one of the most incapacitating side effects of the cancer therapy.[30]
Conclusions Based on these results, we can conclude that the essential oil from Mentha x villosa leaves possesses significant cytotoxic and antitumour activity with low systemic toxicity. It is possible that these actions of the EOMV are related to the synergistic action of its minor constituents. Therefore, further investigations to elucidate the mechanisms of the cytotoxic and antitumour effects exhibited are required.
Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.
Funding JMB-F, DPdS, MOM, COP and SMT are recipients of CNPq productivity grants.
Acknowledgements Authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC/SE).
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25. Kondo A et al. Epigallocatechin-3gallate potentiates curcumin’s ability to suppress uterine leiomyosarcoma cell growth and induce apoptosis. Int J Clin Oncol 2013; 18: 380–388. 26. Lee YL et al. Oral administration of Agaricus blazei (H1 strain) inhibited tumor growth in a sarcoma 180 inoculation model. Exp Anim 2003; 52: 371–375. 27. Cao X et al. Augmentation of hematopoiesis by fibroblast-mediated interleukin-6 gene therapy in mice with chemotherapy. J Interferon Cytokine Res 1998; 18: 227–233. 28. Saif MW. Secondary hepatic resection as a therapeutic goal in advanced colorectal cancer. World J Gastroenterol 2009; 15: 3855–3864. 29. Zamagni C et al. The combination of paclitaxel and carboplatin as first-line chemotherapy in patients with stage III and stage IV ovarian cancer: a phase I-II study. Am J Clin Oncol 1998; 21: 491–497. 30. Takiguchi N et al. Use of 5-FU plus hyperbaric oxygen for treating malignant tumors: evaluation of antitumor effect and measurement of 5-FU in individual organs. Cancer Chemother Pharmacol 2001; 47: 11–14.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web site: Appendix S1 The spectroscopic data of rotundifolone.
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1100–1106
Research Paper
Journal of Pharmacy And Pharmacology
Evaluation of the cytotoxic and antitumour effects of the essential oil from Mentha x villosa and its main compound, rotundifolone Ricardo G. Amarala, Cecília S. Fonsecab, Tayane Kayne M. Silvab, Luciana N. Andradea, Maria E. Françac, José M. Barbosa-Filhod, Damião P. de Sousad, Manoel O. Moraesc, Cláudia Ó Pessoac, Adriana A. Carvalhob and Sara Maria Thomazzia a
Department of Physiology, Federal University of Sergipe, São Cristóvão, bDepartment of Pharmacy, Federal University of Sergipe, Lagarto, Sergipe, Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará and dDepartment of Pharmaceutical Sciences, Federal University of Paraíba, João Pessoa, Paraíba, Brazil c
Keywords antitumour; cytotoxic; essential oil; Mentha x villosa; terpenes Correspondence Sara Maria Thomazzi, Department of Physiology, Federal University of Sergipe, 49100-000, São Cristóvão (SE), Brazil. E-mail: [email protected] Received November 24, 2014 Accepted February 8, 2015 doi: 10.1111/jphp.12409
Abstract Objectives The aim of this study was to investigate the cytotoxic and antitumour effects of the essential oil from the leaves of Mentha x villosa (EOMV) and its main component (rotundifolone). Methods In-vitro cytotoxic activity of the EOMV and rotundifolone was determined on cultured tumour cells. In-vivo antitumour activity of the EOMV was assessed in sarcoma 180-bearing mice. Key findings The EOMV displayed cytotoxicity against human tumour cell lines, showing IC50 values in the range of 0.57–1.02 μg/ml in the HCT-116 and SF-295 cell lines, respectively. Rotundifolone showed weak cytotoxicity against HCT-116, SF-295 and OVCAR-8 cell lines (IC50 > 25.00 μg/ml). Tumour growth inhibition rates were 29.4–40.5% and 25.0–45.2% for the EOMV treatment by intraperitoneal (50–100 mg/kg/day) and oral (100–200 mg/kg/day) administration, respectively. The EOMV did not significantly affect body mass and macroscopy of the organs. Conclusions The EOMV possesses significant antitumour activity with low systemic toxicity, possibly due to the synergistic action of its minor constituents.
Introduction Cancer is a leading cause of death worldwide, accounting for 8.2 million deaths in 2012 with 14.1 million new cases.[1] An exceptionally difficult problem in cancer treatment is multidrug resistance, when cancer cells lose their sensitivity to multiple structurally different chemotherapeutics, leading to the search for alternative treatments, such as medicinal plants. Several studies have demonstrated anticancer activity for the essential oils obtained from medicinal plants.[2–4] Some essential oils of the genus Mentha, an important member of the family Lamiaceae, have been described in the literature for its cytotoxic properties, such as Mentha spicata L. and Mentha arvensis L.[5,6] The species Mentha x villosa Hudson, popularly known as ‘hortelã-da-folha-miúda’ or ‘hortelã-rasteira’, is an herb 1100
largely cultivated in northeastern Brazil, where it has widespread use as a condiment for meat and pasta, and as a salad ingredient.[7] This species also possesses ethnobotanical use in the treatment of worm, influenza, inflammation and pains in general, fever, intestinal infection, indigestion, sinusitis, ophthalmic problems, otalgia, menstrual colic, headache, migraine, and stroke.[8] The literature suggests that the essential oils obtained from Mentha x villosa have various biological activity, such as antinociceptive, hypotensive, spasmolytic, schistosomicidal, antimicrobial and larvicidal.[9–14] Rotundifolone (Figure 1), also known as piperitenone oxide, is a naturally occurring monoterpenic ketone of plant origin and an important chemical constituent of the essential oils of many Mentha species: Mentha rotundifolia
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Antitumour activity of M. x villosa essential oil
O
O
Figure 1 Chemical structure of rotundifolone (1,2-epoxy-p-menth4(8)-en-3-one).
(L.) Huds., Mentha suaveolens Ehrh., Mentha longifolia (L.) Huds., M. spicata and M. x villosa.[15,16] Previous studies have demonstrated that this compound has similar activity to those found in the essential oil of M. x villosa as hypotensive, larvicidal, antinociceptive and schistosomicidal.[9,10,12,14,16] However, there are no studies about the cytotoxic and antitumour activity of the essential oil of M. x villosa and its main constituent. Based on the considerations above, we decided to investigate the effects of the essential oil of Mentha x villosa leaves (EOMV) and rotundifolone, assessing their cytotoxic or antitumour actions, as well as the systemic toxicity of the EOMV.
Materials and Methods
tial oil was submitted to preparative thin layer chromatography. The plates were developed three times using hexanes as eluent. When the plates were exposed to UV light (254 nm), rotundifolone was visualized as the major component of the essential oil. Rotundifolone was removed from the chromatographic plates and later recovered by means of extraction with dichloromethane, followed by filtration and evaporation to obtain a yellowish oil. The structural identification was made with infrared, 1H and, 13C nuclear magnetic resonance analysis, and in comparison with the literature data.[15]
Animals A total of 80 Swiss mice (females, 25–30 g), obtained from the central animal house of the Federal University of Sergipe, Brazil, was used. Animals were housed in cages with free access to food and water. All animals were kept under a 12:12 h light–dark cycle. Animals were treated according to the ethical principles for animal experimentation of the SBCAL (Brazilian Association of Laboratory Animal Science), Brazil. The Animal Studies Committee from the Federal University of Sergipe approved the experimental protocol (CEPA/UFS 24/2013).
Plant material
Cells
M. x villosa leaves were collected in the Medicinal Plant Garden of the Pharmaceutical Technology Laboratory of the Federal University of Paraíba, Brazil, in July 2008 (07°08′29″S, 34°50′48″W). The plant was authenticated and a voucher specimen has been deposited at the Herbarium Prisco Bezerra of the Federal University of Ceará, Brazil (Number 14996).
The cytotoxicity of the EOMV and rotundifolone was tested against OVACAR-8 (ovarian adenocarcinoma), HCT-116 (colon carcinoma) and SF-295 (glioblastoma) human cancer cell lines, all obtained from the National Cancer Institute, Bethesda, MD, USA. Cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, and were then incubated at 37°C in a 5% CO2 atmosphere. Sarcoma 180 tumour cells, which had been maintained in the peritoneal cavity of Swiss mice, were obtained from the Laboratory of Experimental Oncology at the Federal University of Ceará, Brazil.
Extraction of the essential oil Fresh leaves of Mentha x villosa (10 kg) were submitted to hydrodistillation for 8 h using a Clevenger-type apparatus, and an oil of yellowish colouration and characteristic odour of mint was obtained. Subsequently, the oil was dried over anhydrous sodium sulfate, filtered and stored at 4°C. The percentage of yield of the EOMV was 0.1%.[14] The same batch of the EOMV tested in this manuscript was analysed through gas chromatography-mass spectrometry (GC-MS), and the results were described by Lima et al.[14] The essential oil of Mentha x villosa leaves contained rotundifolone (70.96%), limonene (8.75%), germacrene D (3.81%), myrcene (3.10%), trans-caryophyllene (1.46%) and other constituents in a low amount.
Isolation of rotundifolone Rotundifolone was isolated from the EOMV using a procedure previously described by Almeida et al.[15] The essen-
In-vitro cytotoxic activity assay Tumour cell growth was determined by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product.[17] For all experiments, cells were seeded in 96-well plates (0.1 × 106 cells/ml in medium, 100 μl). After 24 h, the EOMV and rotundifolone obtained from the EOMV (0–25 μg/ml), dissolved in 0.07% DMSO, were added to each well (three independent experiments, performed in triplicate). Afterwards, the cells were incubated for 72 h at 37°C in a 5% CO2 atmosphere. Doxorubicin (0.003–0.25 μg/ml) was used as positive control. At the end
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of the incubation, the plates were centrifuged, and the medium was replaced by fresh medium (150 μl) containing 0.5 mg/ml MTT. Three hours later, the formazan product was dissolved in 150 μl DMSO, and absorbance was measured at 595 nm using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc., Fullerton, CA, USA).
In-vivo antitumour activity assay The in-vivo antitumour effect was evaluated using sarcoma 180 ascites tumour cells, following the protocol previously described, with minor modifications.[2] Ten-day-old sarcoma 180 ascites tumour cells (2 × 106 cells per 500 μl) were implanted subcutaneously into the right axillary region of mice. After 24 h, the essential oil was dissolved in 10% DMSO (vehicle) and administered intraperitoneally (50 or 100 mg/kg) or orally by gavage (100 or 200 mg/kg) once a day for seven consecutive days. At the beginning of the experiment, the mice were divided into groups (n = 10 animals/group): (1) intraperitoneal injection of vehicle; (2) intraperitoneal injection of 5-Fluorouracil (5-FU, 25 mg/kg/day); (3) intraperitoneal injection of the EOMV at 50 mg/kg/day; (4) intraperitoneal injection of the EOMV at 100 mg/kg/day; (5) the oral route (p.o., gavage) with vehicle; (6) gavage (p.o.) with cyclophosphamide (10 mg/ kg/day); (7) gavage (p.o.) with the EOMV at 100 mg/kg/ day; and (8) gavage (p.o.) with the EOMV at 200 mg/kg/ day. On day 8, under isoflurane inhalation (1.5%, calibrated vaporizer) anaesthesia, the animals were euthanized by cervical dislocation, and the tumours were removed and weighed. The inhibition ratio of tumour growth (%) was calculated by the following formula: inhibition ratio (%) = ((A – B) / A) × 100, where A is the average tumour weight of the vehicle group and B is the average tumour weight of the treated group.[2]
Systemic toxicological analysis Body mass loss, organ weight alterations and haematological analysis were determined at the end of the experiment above.[2] Body weights were determined at the
start and on the last day of treatment. Before euthanasia, 24 h after the last treatment day, peripheral blood samples of the mice were collected from the retro-orbital plexus under isoflurane inhalation (1.5%, calibrated vaporizer) anaesthesia. For haematological analysis, an aliquot of blood from each animal was placed in EDTA, and haematological parameters (total and differential leucocyte counts) were determined by standard manual procedures using light microscopy. After euthanasia, the livers, spleens and kidneys were dissected and weighed. The wet mass of each organ was expressed as grams per 100 g of body mass and compared with the vehicle group.
Statistical analysis Data are presented as mean ± standard error of mean or half maximal inhibitory concentration (IC50) values, and their 95% confidence intervals were obtained through nonlinear regression. The differences between experimental groups were compared using analysis of variance, followed by the Student–Newman–Keuls test (P < 0.05).
Results The cytotoxic activity of the EOMV and rotundifolone was evaluated against several human tumour cell lines: HCT116 (colon), OVCAR-8 (ovarian) and SF-295 (brain). The IC50 values are shown in Table 1. The EOMV displayed cytotoxicity against all tumour cell lines tested, showing IC50 values in the range of 0.57–1.02 μg/ml in the HCT116 and SF-295 cell lines, respectively. Rotundifolone showed IC50 values > 25.00 μg/ml in all tumour cell lines tested. Doxorubicin, used as the positive control, showed IC50 values ranging from 0.01 to 1.20 μg/ml for HCT-116 and OVCAR-8 cell lines, respectively. The next step of this study was the evaluation of the in-vivo antitumour activity of the EOMV. The effects of the EOMV on mice transplanted with sarcoma 180 tumour cells are presented in Figure 2. One day after the end of the treatment, the average tumour weight of the control (vehicle) mice was 1.90 ± 0.15 and 1.04 ± 0.07 g for intraperitoneal and oral administration, respectively (Figure 2).
Table 1 Cytotoxic activity of the essential oil from the leaves of Mentha x villosa (EOMV) and its main component (rotundifolone) on cancer cell lines Cell lines (IC50 values (μg/ml)) Treatment
HCT-116
OVCAR-8
SF-295
EOMV Rotundifolone Doxorubicin
0.57 (0.44–0.75) >25.00 0.01 (0.01–0.02)
0.86 (0.78–0.96) >25.00 1.20 (0.90–1.60)
1.02 (0.92–1.14) >25.00 0.24 (0.17–0.36)
Cell lines: HCT-116 (human colon carcinoma), OVCAR-8 (human ovarian adenocarcinoma) and SF-295 (human glioblastoma). Data are presented as IC50 values in μg/ml and their 95% confidence interval from three independent experiments, performed in triplicate, and measured by the MTT assay after 72 h of incubation.
1102
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60
1.2 40
0.8
20
0.4
0
0.0 Vehicle
Cyclo
100
200
EOMV (mg/kg/day, p.o.) Figure 2 The effect of the essential oil from the leaves of Mentha x villosa on mice inoculated with sarcoma 180 tumour cells. The graphs show tumour mass (g) and tumour growth inhibition levels (%). The animals were treated by intraperitoneal (a) or oral (b) administration, starting 1 day after tumour implantation, for seven consecutive days. 5-Fluorouracil (5-FU, 25 mg/kg/day, i.p.) and cyclophosphamide (Cyclo, 10 mg/kg/day, p.o.) were used as the positive controls. Vehicle (10% DMSO) was used as the negative control. Data are presented as mean ± standard error of mean (n = 10 animals/group). *P < 0.05 compared with the vehicle group using analysis of variance, followed by the Student–Newman–Keuls test.
In the presence of the EOMV (50 or 100 mg/kg/day) by intraperitoneal administration, the average tumour weights were 1.34 ± 0.16 and 1.13 ± 0.15 g, respectively. In the presence of the EOMV (100 or 200 mg/kg/day) by oral administration, the average tumour weights were 0.78 ± 0.11 and 0.57 ± 0.11 g, respectively. Tumour growth inhibition rates were 29.4–40.5% and 25.0–45.2% for the EOMV treatment by intraperitoneal and oral administration, respectively. In both routes of administration, there were statistically significant differences between both doses in relation to the vehicle group (P < 0.05). 5-FU (25 mg/kg/day, i.p.) and cyclophosphamide (10 mg/kg/day, p.o.) reduced tumour mass by 69.4% and 59.5%, respectively. Systemic toxicological parameters were also examined in the EOMV-treated mice as shown in Tables 2 and 3. No sig© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1100–1106
1.25 ± 0.04 1.11 ± 0.06 1.30 ± 0.06 1.24 ± 0.04 0.52 ± 0.02 0.47 ± 0.03 0.60 ± 0.04 0.41 ± 0.04
1.22 ± 0.04 1.26 ± 0.06 1.18 ± 0.04 1.30 ± 0.03
Sarcoma 180 tumour cells (2.0 × 106cells/animal, s.c.) were implanted in mice. The animals were treated, starting 1 day after tumour implantation, for seven consecutive days. Data are presented as mean ± standard error of mean (n = 10 animals/group). *P < 0.05 compared with the vehicle group using analysis of variance, followed by the Student–Newman–Keuls test.
1.6
Cyclophosphamide
80
Inhibition (%)
Tumour mass (g)
2.0
100
5.06 ± 0.11 4.91 ± 0.31 5.65 ± 0.31 5.16 ± 0.16
Inhibition
0.9 ± 0.3 0.6 ± 0.3 0.3 ± 0.2 −0.4 ± 0.3
Tumour mass
– 100 200 10
(b) 2.4
5-Fluorouracil Oral route Vehicle (10% DMSO) EOMV
EOMV (mg/kg/day, i.p.)
0.58 ± 0.04 0.61 ± 0.03 0.56 ± 0.03 0.36 ± 0.03*
100
4.83 ± 0.25 4.72 ± 0.19 4.71 ± 0.15 4.99 ± 0.16
50
2.1 ± 0.4 0.9 ± 0.4 0.7 ± 0.6 −3.1 ± 0.5*
5-FU
50 100 25
0 Vehicle
–
0.0
Intraperitoneal route Vehicle (10% DMSO) EOMV
20
0.4
Spleen (g/100 g body mass)
40
0.8
Liver (g/100 g body mass)
60
1.2
The effect of the essential oil from the leaves of Mentha x villosa (EOMV) on body and organ weights of mice transplanted with sarcoma 180 tumour cells
1.6
Variation in body mass (g)
80
Inhibition (%)
Tumour mass (g)
2.0
100
Dose (mg/kg/day)
Inhibition
Treatment
Tumour mass
Table 2
(a) 2.4
Antitumour activity of M. x villosa essential oil
Kidney (g/100 g body mass)
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Table 3
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The effect of the essential oil from the leaves of Mentha x villosa (EOMV) on the haematological parameters in peripheral blood
Treatment Intraperitoneal route Vehicle (10% DMSO) EOMV 5-Fluorouracil Oral route Vehicle (10% DMSO) EOMV Cyclophosphamide
Differential count of leucocytes (%)
Dose (mg/kg/day)
Total leucocytes (103 cells/μl)
Eosinophil
Lymphocyte
Neutrophil
– 50 100 25
8.8 ± 0.5 7.3 ± 0.5*# 6.2 ± 0.2*# 2.4 ± 0.4*
0.4 0.4 0.2 0.2
50.4 54.2 57.2 75.2*
44.2 39.6 37.0 21.2*
5.0 5.8 4.2 3.6
– 100 200 10
9.5 ± 1.1 8.3 ± 0.7 7.7 ± 2.6 6.3 ± 0.8
0.2 0.2 0.0 0.0
59.3 55.6 60.4 61.2
32.5 36.6 31.6 27.8
8.0 7.6 8.0 11.0
Monocyte
Sarcoma 180 tumour cells (2.0 × 106cells/animal, s.c.) were implanted in mice. The animals were treated, starting 1 day after tumour implantation, for seven consecutive days. Data are presented as mean ± standard error of mean and % (n = 10 animals/group). *P < 0.05 compared with the vehicle group and #P < 0.05 compared with the 5-fluorouracil group by analysis of variance, followed by Student–Newman–Keuls.
nificant changes were observed in body mass gain after administration of the EOMV by both routes (P > 0.05, Table 2). Also, no significant changes in the mass of livers, kidneys or spleens were seen in the essential oil-treated groups (P > 0.05). Cyclophosphamide also failed to significantly change the body mass and the mass of organs, compared with the vehicle group (10% DMSO). In contrast, 5-FU reduced the body mass and spleen organ mass (P < 0.05). Another systemic toxicological parameter examined was the blood leucocytes count, as shown in Table 3. In the peripheral blood from mice transplanted with sarcoma 180 tumour cells, the EOMV and 5-FU induced a decrease in total leucocytes (P < 0.05) after intraperitoneal administration. However, leucopenia was more pronounced in 5-FUtreated animals compared with the EOMV-treated groups (50 and 100 mg/kg/day, i.p.; P < 0.05). Different from the results observed after intraperitoneal administration, the EOMV and cyclophosphamide reduced tumour development (Figure 2) without inducing a disturbance in the leucocytes count when administered orally (Table 3).
Discussion In the present work, we demonstrate for the first time the in-vitro cytotoxic activity of the EOMV and its main component (rotundifolone) against tumour cell lines, and the in-vivo antitumour effect of the EOMV on mice transplanted with sarcoma 180 tumour cells as well as its systemic toxicity. Previous GC-MS performed by Lima et al. with the same batch of the essential oil used in this study showed the presence of rotundifolone (70.96%), limonene (8.75%), germacrene D (3.81%), myrcene (3.10%) and transcaryophyllene (1.46%) as major compounds.[14] Interest1104
ingly, the major component in the EOMV identified by other authors was also rotundifolone, despite variations in the percentage composition (58.74–95.87%).[12,18,19] Generally, the major components are found to reflect quite well the biophysical and biological features of the essential oils from which they were isolated, the amplitude of their effects being just dependent on their concentration when they were tested alone. However, it is possible that the activity of the main components is modulated by other minor components.[20,21] Therefore, besides the EOMV, we investigated the cytotoxic action of rotundifolone on cancer cell lines. According to the preclinical anticancer drug screening programme used in this study, an essential oil that shows IC50 values below 30 μg/ml and an isolated compound that shows IC50 values below 1 μg/ml are considered promising.[3,4] Therefore, we considered that the EOMV presents potent cytotoxic activity, and rotundifolone obtained from Mentha x villosa plant has weak cytotoxic activity against the tumour cell lines tested in the concentration range used. The potent cytotoxic activity of the EOMV demonstrated in this study might be attributed to the mixture of its other constituents. Many studies have demonstrated that several constituents found in the EOMV, including sabinene, β-pinene, myrcene, limonene and germacrene D show cytotoxic and anticancer activity justifying the in-vitro effect shown by the EOMV and its possible action in vivo.[20,22,23] Previous studies have demonstrated the synergistic action of the combination of natural compounds with anticancer activity. Rahman et al. demonstrated that the combination of two plant bioactive compounds, epigallocatechin gallate (a catechin) and [6]-gingerol (a major component of ginger), synergistically induces apoptosis and inhibits the proliferation of 1321N1 and LN18 cells (human glioblastoma cell lines).[24] The combination treatment of epigallocatechin gallate and curcumin (a phenolic
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compound) showed synergistically cytotoxic action towards uterine leiomyosarcoma cells in vitro, and it reduced uterine leiomyosarcoma cells viability more than did the treatment with either drug alone.[25] Sarcoma 180 is an original mouse tumour, transplantable and well-characterized experimental model used in the research of in-vivo antitumour activity.[26] Currently, most of the drugs used in chemotherapy are administered by the parenteral route. However, there are few drugs that can be used by the oral route. Therefore, we decided to evaluate the antitumour activity of the EOMV using two different routes of administration: intraperitoneal (a parenteral route) and oral/gavage (an enteral route). The EOMV showed antitumour activity with both administration routes (i.p. and p.o.), but it was more potent by intraperitoneal administration. In fact, usually, the oral route is disadvantageous because of less absorption. Ingested drugs are subject to the first-pass effect, in which a significant amount of the agent is metabolized in the gut wall and the liver before it reaches systemic circulation. Thus, some drugs have low bioavailability when given orally.[2] Rotundifolone was not tested in an animal-bearing tumour due to its weak cytotoxic activity against the tumour cell lines. Most cancer chemotherapeutic agents are non-targeted in action and demolish rapidly dividing normal cells besides tumours, leading to extensive side effects. Some of the common clinical side effects include hepatic dysfunction, renal toxicity and haematopoietic depression. The liver plays a major role in metabolism and the kidney in the urinary system, and both have a number of functions in the body, including mostly detoxification and excretion of wastes, respectively. On the other hand, the spleen is an organ with important roles regarding the red blood cells and the immune system. Hepatic dysfunction induced by irinotecan, renal toxicity induced by docetaxel and haematopoietic suppression induced by 5-FU are such examples.[27–29] In addition, often, vomiting and diarrhoea are observed, leading to loss of body mass and malnutrition. Herein, variation in body mass, hepatotoxic and nephrotoxic effects, as well as changes in haematological parameters, were investigated.
References 1. Ferlay J et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136: E359–E386. 2. Britto ACS et al. In vitro and in vivo antitumor effects of the essential oil from the leaves of Guatteria friesiana. Planta Med 2012; 78: 409–414.
In this study, the treatment with the EOMV did not significantly affect the body mass or macroscopy of the organs (kidney, liver and spleen) by both routes, but induced a decrease in total leucocytes after intraperitoneal administration. Furthermore, we found that unlike the EOMV-treated groups, the treatment with 5-FU decreased the spleen mass, and it induced a leucopenia more pronounced with a disturbance in the differential count of circulating peripheral leucocytes. These results confirm the side effect of this immunosuppressive drug, clinically useful, which substantially increases the risk of infections.[30] This is one of the most incapacitating side effects of the cancer therapy.[30]
Conclusions Based on these results, we can conclude that the essential oil from Mentha x villosa leaves possesses significant cytotoxic and antitumour activity with low systemic toxicity. It is possible that these actions of the EOMV are related to the synergistic action of its minor constituents. Therefore, further investigations to elucidate the mechanisms of the cytotoxic and antitumour effects exhibited are required.
Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.
Funding JMB-F, DPdS, MOM, COP and SMT are recipients of CNPq productivity grants.
Acknowledgements Authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC/SE).
3. Ferraz RPC et al. Antitumour properties of the leaf essential oil of Xylopia frutescens Aubl. (Annonaceae). Food Chem 2013; 141: 196–200. 4. Quintans JSS et al. Chemical constituents and anticancer effects of the essential oil from leaves of Xylopia laevigata. Planta Med 2013; 79: 123– 130. 5. Manosroi J et al. Anti-proliferative activity of essential oil extracted from
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Thai medicinal plants on KB and P388 cell lines. Cancer Lett 2006; 235: 114– 120. 6. Hussain AI et al. Seasonal variation in content, chemical composition and antimicrobial and cytotoxic activities of essential oils from four Mentha species. J Sci Food Agric 2010; 90: 1827–1836. 7. Teles NSB et al. Evaluation of the therapeutic efficacy of Mentha crispa 1105
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web site: Appendix S1 The spectroscopic data of rotundifolone.
© 2015 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 1100–1106
