The essential oils from <i> Mentha viridis </i> (L). L. and <i> Mentha pulegium </i> L. were studied to assess their inhibitory potential on phospholipase from snake venoms and to determine their cytogenotoxic action on huma n cells. These essential oils were able to inhibit the breakdown of phospholipids induced by venoms of snakes of the <i> Bothrops </i> genus. Both oils presented hemolytic activity, although the <i> Mentha viridis </i> (L). L. oil induced hemolysis only at the highest concentrations (14.6 and 29 μL·mL - 1 ). The essential oil from <i> M. viridis </i> induced 3.9; 8.6 and 16.2 times greater damage to human leukocyte DNA than that observed with the positive control (100 μg· μL - 1 doxorubicin) at concentrations of 0.25; 0.5 and 1.0 μL· mL - 1 , respectively. A similar effect was observed for the oil from <i> M. pulegium </i> (2.1 , 2.5 and 15.8 times greater damage). The results extend the characterization of these essential oils and demonstrate their potential use in industries.
Among the many compounds produced by plants, essential oils are volatile materials contained in many plant organs that are related to various functions necessary for plant survival [
Although disclosure of the use of natural products has grown in recent years as described by Bors et al. [
Yamaguchi and Veiga-Junior [
The Mentha, are widely used by humans, with emphasis on the use of essential oils in cosmetics, pharmaceuticals, food, confectionery and industrial alcoholic beverages [
The leaves (rib and limbs) from adult plants of Mentha viridis (L.) L. and Mentha pulegium (L.) species were collected around 07:00 hours on the Campus of the Universidade Federal de Lavras (UFLA) (21˚14'S, longitude 45˚00'W Gr. and 918 m altitude) on days without precipitation in the month of November 2012. Species identification was kindly performed by Dra. Mariana Esteves Mansanares, Department of Biology of UFLA, and exsiccates were deposited in the ESAL Herbarium at UFLA under the registration numbers 27.123 and 27.122, respectively.
The essential oils from fresh leaves were extracted by hydro-distillation using a modified Clevenger apparatus adapted to a 6-L, round-bottom flask over a period of 2 hours. The hydrosols were centrifuged (Fanem Baby I Modelo 206 BL) for 10 minutes at 965g at room temperature, and the oils were packaged in amber glass bottles and stored at a temperature of 4˚C [
The peripheral blood from volunteers aged 21 to 40 years was used after free informed consent; the volunteers did not present disease symptoms and reported not having used prescription drugs for at least 30 days prior to collection. These subjects were randomly selected. This study was approved by the Committee of Ethics in Research with Humans of UFLA and filed under the number 17935713.8.0000.5148.
The concentrations chosen for the evaluation of the essential oils correspond to previously determined non-cytotoxic doses (data not shown). Negative (without treatment) and positive (100 µg∙µL−1 doxorubicin) controls were conducted simultaneously. The blood samples (500 µL) were incubated in an oven at 37˚C in for 4 hours the presence of the treatments. The Comet assay was then performed according to the method described by Singh et al. [
Nucleoids and electrophoresis: An aliquot (20 μL) of each cell suspension containing the treatments was mixed with 100 μL of low-melting-point agarose solution (0.5%wv-1 in Phosphate Buffered Saline-PBS), applied to a microscope slide previously coated with standard normal-melting-point agarose solution (1% w v−1 in PBS), immediately overlaid with a coverslip, and held at 4˚C for 10 minutes. For each treatment/volunteer, three slides were prepared. The coverslips were removed and the slides were immersed in lysis solution (2.5 mol∙L−1 NaCl, 100 mmol∙L−1 EDTA, 10 mmol∙L−1 Tris, 1% Triton X-100, 10% DMSO; pH 10), where they remained for 20 hours at 4˚C to isolate their nucleoids for analysis.
After lysis, the slides were kept in a freshly prepared electrophoresis solution (1 mmol∙L−1 EDTA, 300 mmol∙L−1 NaOH; pH 13) at approximately 15˚C for 25 minutes to expose the alkali-labile sites and then subjected to electrophoresis at 25V for 30 minutes. The slides were kept in a neutralization solution (0.4 mol∙L−1 Tris; pH 7.5) for 25 minutes, dried and fixed with absolute ethanol.
Staining and analysis: The slides were stained with propidium iodide solution (0.5 mg.mL−1), overlaid with a coverslip and analyzed with an epifluorescence microscope (Nikon ECLIPSE E400) at 400× magnifications. To measure damage levels in lymphocyte DNA molecules, 100 nucleoids of each slide and three slides per treatment/volunteer (totalling 300 nucleoids per treatment/volunteer), were counted and classified by the same evaluator using visual score patterns.
The nucleoids were classified according to Collins et al. [
The blood collected (10 mL) in the presence of an anticlotting agent was immediately mixed with saline solution (2 mM NaH2PO4; 3 mM Na2HPO4; 154 mM NaCl; pH 7.4) and centrifuged at 700g (Fanem Baby® IModelo 206 BL) for 5 minutes. The plasma was removed, and the red cells were suspended in 5 mmol∙L−1 phosphate buffer, pH 7.4 and centrifuged under the same conditions. This washing procedure was repeated three times at 4˚C. The 100% red blood cell concentrate was diluted to 2% and 0.15% hematocrits (137.33 and 13 µmol∙L−1 lipid, respectively), using the same buffer [
The hemolytic activity was evaluated by incubating 1 mL of erythrocyte suspension (2% or 0.15%) for 60 minutes at 37˚C with the oil solutions at the concentrations of 0.6; 1.0; 2.0; 3.0; 4.0; 5.0 and 10.0 µL∙mL−1, followed by centrifugation at 1500g for 5 minutes. The hemoglobin concentration was determined in the supernatant by measuring the absorbance at 412 nm (Shimadzu UV-160 1 PC) according to Rangel et al. [
in which Aa, Ac1 and Ac2 are, respectively, the absorbance of the sample and of the controls c1 and c2 at 412 nm.
The hemolysis of human erythrocytes in solid medium was performed according to the method described by Gutiérrez et al. [
The phospholipase activity was evaluated by the use of solid medium, as described by Gutiérrez et al. [
The inhibition of the snake-venom-induced phospholipase activity by the essential oils was also evaluated. Solutions of venom from B. jararaca, B. jararacussu, B. moojeni and B. alternatus (40 μg∙mL−1) were incubated with 5.5; 10.9; 21.8; 43.6 and 87.3 μL∙mL−1 concentrations of the oils at 37˚C for one hour. The gels containing the samples were kept in a cell culture chamber at 37˚C for 24 hours, and the formation of a translucent halo around the hole in the gel characterized phospholipase activity, which was quantified by measuring the halo diameter [
The results of the Comet assay were statistically evaluated by analysis of variance, and the means were compared using the Scott-Knott test (p < 0.05) with the aid of the R software (R Development Core Team, 2011) [
The essential oils from M. viridis and M. pulegium induced DNA fragmentation at all the concentrations evaluated. The highest levels of fragmentation (classes 3 and 4) were observed for M. viridis in all treatments evaluated and for M. pulegium at the concentration of 1 µL∙mL−1.
The arbitrary unit (A.U.) values for the M. viridis treatments were 86.2; 190.0 and 358.4 for doses of 0.25; 0.5 and 1.0 µL∙mL−1, respectively. These values are 3.9; 8.6 and 16.3 times higher than those observed for the negative control (22.0 A.U.), which demonstrates the genotoxic potential of the oils. Treatment with the same concentrations of M. pulegium resulted in A.U. values of 46.6; 55.2 and 349.6, these being 2.1, 2.5 and 15.8 times higher, respectively, than the negative control.
The essential oils from M. viridis and M. pulegium presented A.U. values of 358.4 and 349.6, respectively, when a 1.0 µL∙mL−1 concentration was administered. These values were higher than those observed for the antitumor drug used as a control (159.0), even though this drug was evaluated at a concentration 10 times higher (100 µg∙µL−1) (
The essential oils from M. viridis and M. pulegium, at a concentration of 1 μL∙mL−1, induced percentages of damaged nucleoids (94.2 and 97.0, respectively) higher than that observed with doxorubicin (84.8%).
The major constituents found in the essential oil from Mentha viridis were linalool (40.70%), carvone (13.52%) and α-terpinene (8.56%). The constituents found in the essential oil from Mentha pulegium were pulegone (50.01%), menthol (31.90%) and menthone (16.56%). The highest percentages of totally damaged nucleoids (class 4: damage > 85%) were 16.4; 81.6 and 72.0, respectively, at concentrations of 0.5 and 1.0 µL∙mL−1 for M. viridis and 1.0 µL∙mL−1 for M. pulegium.
With the use of the Comet assay, Péres et al. [
The major constituents present in the oil evaluated by Peres et al. [
Treatment (μL∙mL−1) | Class of Comet (%)1 | Frequency of damage (%)2 | Arbitrary units3 | |||||
---|---|---|---|---|---|---|---|---|
0 | 1 | 2 | 3 | 4 | ||||
Negative control | 78.8 ± 3.4a | 20.2 ± 3.0a | 1.0 ± 0.8a | 0.0 ± 0.0a | 0.0 ± 0.0a | 21.2 ± 3.4a | 22.0 ± 4.2a | |
Positive control4 | 15.2 ± 3.2b | 17.8 ± 3.1a | 36.2 ± 12.4b | 14.8 ± 10.9b | 16.0 ± 3.7b | 84.8 ± 3.2b | 159.0 ± 20.3b | |
M. viridis | 0.25 | 49.8 ± 2.3c | 24.4 ± 2.5a | 18.4 ± 3.1c | 4.6 ± 1.7c | 2.8 ± 2.2a | 50.2 ± 2.3c | 86.2 ± 7.4c |
0.5 | 19.8 ± 6.6b | 9.8 ± 1.9b | 47.4 ± 6.5d | 6.6 ± 3.9c | 16.4 ± 2.1b | 80.2 ± 6.6b | 190.0 ± 17.6d | |
1.0 | 5.8 ± 4.9d | 0.2 ± 0.3c | 5.4 ± 5.3a | 7.0 ± 5.6c | 81.6 ± 6.9c | 94.2 ± 4.9d | 358.4 ± 8.7e | |
M. pulegium | 0.25 | 69.8 ± 2.9e | 16.0 ± 4.4a | 12.6 ± 2.9e | 1.0 ± 0.8a | 0.6 ± 0.9a | 30.2 ± 2.9e | 46.6 ± 4.9f |
0.5 | 63.0 ± 6.8e | 18.8 ± 1.4a | 16.6 ± 8.6c | 1.6 ± 1.9a | 0.0 ± 0.0a | 36.0 ± 6.8e | 55.2 ± 14.1f | |
1.0 | 3.0 ± 3.6d | 0.4 ± 0.6c | 12.6 ± 9.1ab | 12.0 ± 3.2b | 72.0 ± 10.0c | 97.0 ± 3.6d | 349.6 ± 17.9e |
1Data represent the mean values obtained for 300 nucleoids/treatment/volunteer. Five volunteers were used (1 volunteer/experiment); 2Frequency of damage: sum of the damage in classes 1 to 4; 3Arbitrary units: (1 × damage in class 1) + (2 × damage in class 2) + (3 × damage in class 3) + (4 × damage in class 4), according to the method described by Collins et al. (2004) 15; 4Positive control: 100 µg∙µL−1 doxorubicin. The results with the same letters in the columns do not differ by the Scott-Knott test (p < 0.05) in comparison with the controls.
Rassouli et al. [
Despite major advances in cancer therapy, research for new compounds, mainly of natural origin, with potential anticancer and fewer side effects are of great value for the development of new therapies. The results of genotoxicity tests for the essential oils from M. viridis and M. pulegium, whose major constituents are represented by terpenes, emphasize the anticancer potential of these oils when compared to the allopathic drug doxorubicin.
The essential oil from M. pulegium presented an increase in hemolytic activity starting at a concentration of 5.5 μL∙mL−1 and ending at 87.3 μL∙mL−1, considering limitations of the volumes implicit to each method employed. Activity was observed only at the highest concentrations (43.6 and 87.3 μL∙mL−1) of the oil from M. viridis (
During tests of inhibition of L. muta venom, a greater activity was observed for the essential oil from M. viridis at a concentration of 21.8 μL∙mL−1 (
Nearly 100% hemolytic activity was observed for all the concentrations (0.6 to 10 µL∙mL−1) of the two essential oils tested in liquid medium at 0.15% hematocrit
(
Constituents present in oils may interact with components of erythrocyte membranes, leading to destabilization of its structure and a disorderly influx of ions and water that result in rupture of the membranes. Thus, the reduction in the number of erythrocytes in solution (0.15% hematocrit) probably resulted in interactions between membrane structures and constituents present in oils and increased the hemolytic effects observed for both oils. However, a smaller number of such molecules present in the oils in the presence of a larger number of cells (2% hematocrit) can promote interactions with proteins and membrane lipids without altering their conformations, resulting in a protective effect against the action of other molecules such as enzymes with phospholipase or proteolytic activity present in venoms. The human organism has a hematocrit of approximately 45%, much higher than those used during the tests. In addition, the erythrocytes are not isolated, but rather dispersed among several other cell types, proteins, lipids, carbohydrates, ions etc., which could lead to unpredictable effects for the essential oils evaluated in this study.
Marya et al. [
cloves and the principal constituents, eugenol and eugenyl acetate, in human erythrocytes (10% hematocrit). At the highest concentration tested (0.2% v/v), eugenyl acetate, eugenol and the essential oil of cloves induced 57; 40 and 48% hemolysis, respectively. The authors showed that an increase in the concentration of the essential oil and the major constituents resulted in a higher hemolytic activity similar to the effect observed in this study. The major constituents of the essential oils of M. pulegium and M. viridis are sesquiterpenes, and those in the essential oil of cloves are phenylpropanoids. However, both essential oils were effective in inducing high hemolytic activity.
Quintans et al. [
Misharina et al. [
A 20.6% inhibition of the phospholipase activity of B. jararaca (
We observed a 100% inhibition of phospholipase activity induced by B. alternatus in the tests with the oil from M. pulegium at concentrations of 21.8 and 43.6 μL∙mL−1. A decrease in the inhibitory effect (inhibition close to 40%) with increasing concentration of the oil (87.3 μL∙mL−1) (
Terra and Lema [
Fernandes et al. [
The inhibitory effect of the extract was dose dependent, whereas the isolated compound was able to inhibit 50% of the activity of the venoms at the concentration of 300 μL∙mL−1.
The inhibitory effect of arjunolic acid is superior to those observed for M. pulegium (approximately 20.6% inhibition of the activity of B. jararaca venom and 4.0% inhibition of B. jararacussu venom) and M. viridis oils (no effect on the activity of B. jararaca venom and approximately 4.0% inhibition of B. jararacussu venom). Although, several biological activities exhibited by plant extracts or essential oils are assigned to sets of molecules that act in synergy, various activities involve isolated compounds that interact with animal enzymes.
Strauch et al. [
The inhibitory effect on phospholipase suggests a possible anti-inflammatory potential for the essential oils. The cleavage of phospholipids by phospholipase A2 present in venoms can be compared to the phospholipase activity exerted by endogenous enzymes in mammals. This specific enzymatic activity results in the breakdown of membrane phospholipids to release fatty acids and lysophospholipids, including arachidonic acid, precursor of bioactive lipids (e.g. prostaglandins, leukotrienes and thromboxanes) that operate in various physiological processes, including inflammatory and immune responses [
The essential oils from M. pulegium and M. viridis presented a high genotoxic potential even when evaluated at lower doses. Considering the limited options for chemotherapy available today, the oils evaluated showed promise for alternative pharmaceutical formulations. In addition, they have a potential for use as adjuvants in the treatment of snake bites and as sources of active principles for the development of anti-inflammatory or immune response modulators.
The authors acknowledge the support of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a PVNS.
The authors declare no conflict of interest.
Silva, L.F., das Graças Cardoso, M., Preté, P.S.C., Teixeira, M.L., Nelson, D.L., Magalhães, M.L., Ferreira, V.R.F., Souza, R.V., Soares, L.I. and Marcussi, S. (2017) Essential Oils from Mentha viridis (L). L. and Mentha pulegium L.: Cytogenotoxic Effects on Human Cells. American Journal of Plant Sciences, 8, 1423-1437. https://doi.org/10.4236/ajps.2017.86097