|Year : 2017 | Volume
| Issue : 49 | Page : 114-117
Effects of insecticidal ketones present in mint plants on GABAA receptor from mammalian neurons
Mariela Eugenia Sánchez-Borzone, Leticia Delgado Marin, Daniel Asmed García
Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT), CONICET-Universidad Nacional de Córdoba, Av. Vèlez Sarsfield 1611, Córdoba 5016, Argentina
|Date of Submission||31-Jan-2016|
|Date of Acceptance||08-Feb-2016|
|Date of Web Publication||06-Jan-2017|
Daniel Asmed García
Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT), CONICET-Universidad Nacional de Córdoba, Cátedra de Química Biológica, FCEFYN, Av. Vélez Sarsfield 1611,Córdoba 5016
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The genus Mentha, an important member of the Lamiaceae family, is represented by many species commonly known as mint. The insecticidal activity of Mentha oil and its main components has been tested and established against various insects/pests. Among these, the ketone monoterpenes that are most common in different Mentha species demonstrated insect toxicity, with pulegone being the most active, followed by carvone and menthone. Considering that the GABAA receptor (GABAA-R) is one of the main insecticide targets on neurons, and that pulegone would modulate the insect GABA system, it may be expected that the insecticidal properties of Mentha ketones are mediated by their interaction with this receptor. Objective: In order to discern the pharmacological actions of these products when used as insecticides on mammalian organisms, we evaluated the pharmacologic activity of ketones, commonly present in Mentha plants, on native GABAA-R from rats. Materials and Methods: Determination of ketones effects on allosterically enhanced benzodiazepine binding, using primary cultures of cortical neurons, which express functional receptors and MTT assay to evaluate their cell toxicity. Results: Our results seem to indicate that ketone components of Mentha, with proven repellent or insecticide activity, were able to behave as GABAA-R negative allosteric modulators in murine cells and consequently could exhibit convulsant activity in mammalians. Only pulegone at the highest assayed concentration (2 mM) showed a significant reduction in cell viability after exposure for 24 hr. Conclusion: The present results strongly suggest that the ketone components of Mentha are able to exhibit convulsant activity in mammalian organisms, but functional assays and β€in vivo experiments would be necessary to corroborate this proposed action.
Abbreviations used: GABA: gamma aminobutyric acid, GABAA-R: GABAA receptor, MTT: 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazam, DMEM: Dulbecco's modified minimum essential mèdium, [3H]TBOB: [3H] t-Butylbicycloorthobenzoate
Keywords: Cell culture, GABAA receptor, insecticide, ketones, Mentha, toxicity
|How to cite this article:|
Sánchez-Borzone ME, Marin LD, García DA. Effects of insecticidal ketones present in mint plants on GABAA receptor from mammalian neurons. Phcog Mag 2017;13:114-7
|How to cite this URL:|
Sánchez-Borzone ME, Marin LD, García DA. Effects of insecticidal ketones present in mint plants on GABAA receptor from mammalian neurons. Phcog Mag [serial online] 2017 [cited 2017 Sep 26];13:114-7. Available from: http://www.phcog.com/text.asp?2017/13/49/114/197638
- The pharmacological activity of insecticide ketones, commonly present in Mentha plants, was evaluated on native GABAA receptor from mammalian neurons.
- All studied compounds: pulegone, menthone and dihydrocarvone, were able to behave as negative allosteric modulators and could exhibit convulsant activity in mammalian organisms.
- Citotoxicity assays demonstrated that only pulegone affected the cell viability.
| Introduction|| |
The genus Mentha, one of the important members of the Lamiaceae family, is represented by many species commonly identified as mint, which has been known for its medicinal and aromatherapy properties. The insecticidal activity of Mentha oil and its main components has been tested and established against various insects/pests. Mentha's repellent properties against agricultural pests were investigated in a series of experiments by Odeyemi et al. and Kumar et al. Its repellent activity was also demonstrated in mosquito control, and thus for diseases of public health concern such as malaria, yellow fever, dengue, and viral encephalitis.– Many assays have reported insect mortality caused by Mentha toxicity,, and some have evaluated its antifeedant activity., Species of the genus Mentha have been reported to contain a range of components. The monocyclic ketones most commonly found in Mentha species are pulegone, menthone, carvone, and, to a lesser extent, dihydrocarvone.,
The GABAA receptor (GABAA-R) is a major insecticide target along with the voltage-dependent sodium channel, the nicotinic receptor, and acetylcholinesterase., Important insecticides acting at the GABAA-R (e.g., lindane, α-endosulfan, dieldrin, and fipronil) recognize the picrotoxinin site, a noncompetitive antagonist site, to block GABA-induced chloride flux. GABAA-R in mammalian, and even in various insect species, differs a lot in their subunit combinations and sensitivities to different ligands.,, The structure and nature of binding sites in housefly GABA receptors have been shown to be different from those in rat GABA receptors, and the differences may be related to the selectivity of antagonists for housefly versus rat receptor.
We described very recently the effects of carvone isomers on the mammalian GABAA-R, demonstrating their inhibitory activity on this receptor. In this work, we evaluated the pharmacologic activity of the other monoterpene ketones commonly present in Mentha (pulegone, menthone, and dihydrocarvone; see structures in [Figure 1]) on native GABAA-R from rats by determining their effects on allosterically enhanced benzodiazepine binding using primary cultures of cortical neurons, which express functional receptors,, in order to discern the pharmacologic activity of these products on mammalian organisms when used as insecticides. We also investigated the possible neurotoxic effects of Mentha components in the same cell culture system at concentrations relevant to their neuroactive ranges.
| Material and Methods|| |
(R)-(+)-Pulegone (purity 99%) (IUPAC name: (5R)-5-methyl-2-propan-2-ylidenecyclohexan-1-one), (–)-menthone (purity 99%) (IUPAC name: (2S,5R)-5-methyl-2-propan-2-ylcyclohexan-1-one), (+)-dihydrocarvone (mixture of isomers: ~77% n-(+)dihydrocarvone and ~20% iso-(+)dihydrocarvone) (IUPAC name: 2-methyl-5-prop-1-en-2-ylcyclohexan-1-one) , γ-aminobutyric acid (GABA), picrotoxin, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazam (MTT), Dulbecco's modified minimum essential medium (DMEM), trypsin, soybean trypsin inhibitor, DNase, amino acids, and poly-l-lysine were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal calf serum was obtained from PAA (Pasching, Austria) and Hflunitrazepam (84.9 Ci/mmol) from Perkin-Elmer (Boston, USA). All the other chemicals were of analytical grade.
Primary cultures of cortical neurons were prepared from the cerebral cortices of 17-day-old rat fetuses, as previously described. The cell suspension (1.6 × 106 cells/ml) was seeded in 24×- or 96× multiwell plates, according to the experiment, precoated with poly-l-lysine and finally incubated for 6-7 days in a humidified 5% CO2/95% air atmosphere at 37°C. Twenty millimolar cytosine arabinoside was added after 48 h in culture to prevent glial proliferation.
The benzodiazepine binding to intact cultured cortical neurons was determined as previously described using nearly 2.0 nM [3H]flunitrazepam. Seven hundred fifty micromolar of ketones and variable concentrations of GABA, between 0 and 200 μM, were added to the incubation media for 30 min of incubation at 25 °C. Nonspecific binding was determined in the presence of 20 μM diazepam.
After 6-7 days in vitro, the cells were exposed to different concentrations of each compound for 30 min or 24 h. Ketones were added after solubilization in 0.2 ml of culture medium previously extracted from each well. Cell viability was determined by measuring the reduction of MTT to a colored formazan salt by mitochondrial reducing activity, as described previously.
Data shown represent the mean ± standard error of mean (SEM). Sigmoid curves were fitted to concentration response data and statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). The two-tailed Student's t-test and one-way ANOVA were used to compare data. A P value less than 0.05 was considered to be statistically significant.
| Results and Discussion|| |
Effects of Mentha ketones on the benzodiazepine binding enhanced by GABA
To evaluate the activity of Mentha ketones on native GABAA-R from mammalians, their effects were observed on [3H]flunitrazepam binding stimulated by the agonist GABA in primary cultures of rat cortical neurons. The results demonstrated that GABA was able to enhance radioligand binding in a dose-dependent manner as expected, showing an EC50 value of 4.4 μM [Figure 2] and [Table 1]. This result is consistent with that reported previously., All Mentha ketones studied in the present work were able to right shift the concentration–response curve of the effect of GABA on [3H]flunitrazepam binding. At the beginning of each curve, pulegone and dihydrocarvone showed a negative effect in the absence of GABA (control samples), while menthone induced an increase; later, all ketones slowly enhanced the binding as the GABA concentration increased. Fitting the data to sigmoid curves revealed a rise in the EC50 value for the GABA-induced increase in [3H]flunitrazepam binding to 123.7, 64.8, and 66.7 μM in the presence of pulegone, menthone, and dihydrocarvone, respectively. At the same time, the maximum response induced by GABA 200 μM (174% with respect to basal) was significantly reduced by all ketones [Figure 2] and [Table 1] (P < 0.05, one-way ANOVA). Taking into account these effects, we can clearly consider all Mentha compounds as negative allosteric modulators, at least on mammalian neurons. It should be noted that the allosteric behavior of the receptor was tested by determining the improvement of [3H]flunitrazepam binding exerted by GABA and its reduction by a noncompetitive GABA antagonist.
|Table 1: Effect of Mentha ketones on GABA-induced increase of [3H]flunitrazepam binding|
Click here to view
Pulegone is used as a flavoring agent, in perfumery and aromatherapy, and an enantiomeric form ((R)-(+)-pulegone) was described as a psychoactive compound with the profile of an analgesic drug. Tong and Coats suggested that pulegone and other monoterpenoids act as positive allosteric modulators of the GABAA-R in insects. However, this result supported by the 36Cl– uptake enhancement did not correlate very well with the increase found in [3H]TBOB binding in the same work, since a reducing effect should be expected according to their suggested function as GABA allosteric agonist. The inhibitory effect of pulegone on GABA-stimulated [3H]flunitrazepam binding described in the present work clearly indicates its activity as a negative allosteric modulator in murine cortical neurons. In another report, Bessette described opposite effects of pulegone on [3H]TBOB binding in house fly (enhancement) or in mouse (inhibition). These controversial results could be the consequence of the wide, complex subunit composition of GABAA-R and its different expression in insect compared with mammalian organisms.
Menthone is extensively used in perfumery and as a flavoring agent. The marked increase in the EC50 found in the present study induced by menthone strongly suggests an essentially negative effect on this receptor in mammalian cells. Therefore, considering dissimilar organisms, some differing responses would be expected, as is discussed above with pulegone.
The last Mentha ketone assayed, dihydrocarvone, also showed inhibitory effects on the receptor and could accordingly also be considered an allosteric negative modulator. Even though it was described that hydroxydihydrocarvone, a similar compound, has significant psychopharmacologic activity with depressant effects, this is the first report of the interaction of dihydrocarvone on GABAA-R.
Effects of Mentha ketones on neuron viability
Considering that Mentha essential oils and their principal components have been described as toxic for many insects,,,, we analyzed the toxicity in mammalian cells of three Mentha ketones. Mentha ketones did not reduce cell viability after exposure for 30 min. After 24 h, just pulegone, at the highest tested concentration, showed significant reduction in viability (16%) compared with control (100%) in this cell assay system (P < 0.05, one-way ANOVA). It is interesting to note that the highest toxicity of pulegone, compared with menthone and dihydrocarvone, is in agreement with their reported insect toxicity.,,
Mentha components as repellents or insecticide agents could expose them to other organisms, including mammalian, where their potential toxicity should be evaluated. Pulegone is recognized as potentially toxic to the liver and lung., It has been suggested that its main action mechanism could involve metabolites of pulegone, such as menthofuran, which neutralize cytochrome P450s by modifying the prosthetic heme group or the apoprotein. This mechanism could be occurring in the toxic effect of pulegone on rat neurons at high concentrations and over a long time, as described in the present work. Menthone showed moderate to low acute oral toxicity in rats. In cell viability studies, using a cell line of human foreskin fibroblasts, menthone was found to be nontoxic, while pulegone showed adverse effects. At present, there are no published reports about the cytotoxicity of dihydrocarvone. Its oxidized form, carvone, and its isomer had no effect on murine neuron viability, as we described previously.
In conclusion, the possible use of natural repellents or insecticides based on extracts, essential oils, or their principal components should consider their potential effects on other organisms. The present results seem to indicate that ketone components of Mentha, with proven repellent or insecticide activity, are able to behave as GABAA-R negative allosteric modulators in mammalian cells, although further assays, such as [35S]TBPS and [3H]muscimol binding, would be necessary to identify the exact binding site(s) of the ketones. Thus, the present results strongly suggest that the ketone components of Mentha are able to exhibit convulsant activity in mammalian organisms, but functional assays and in vivo experiments would be necessary to corroborate this proposed action.
This work was partially financed with grants from SECyT-Universidad Nacional de Córdoba, FONCYT (Argentina) and CONICET (Argentina). MSB and DAG are career investigators of CONICET, and LDM holds a fellowship from the same institution
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest
| References|| |
Kumar P, Mishra S, Malik A, Satya S. Insecticidal properties of Mentha
species: a review. Ind Crops Prod 2011;34:802-17.
Odeyemi OO, Masika P, Afolayan AJ. Insecticidal activities of essential oil from the leaves of Mentha longifolia
L. subsp. capensis
against Sitophilus zeamais
(Motschulsky) (Coleoptera: Curculionidae). Afr Entomol 2008;16:220-5.
Kumar A, Shukla R, Singh P, Singh AK, Dubey NK. Use of essential oil from Mentha arvensis
L. to control storage moulds and insects in stored chickpea. J Sci Food Agric 2009;89:2643-9.
Ansari MA, Vasudevan P, Tandon M, Razdan RK. Larvicidal and mosquito repellent action of peppermint (Mentha piperita
) oil. Bioresour Technol 2000;71:267-71.
Erler F, Ulug I, Yalcinkaya B. Repellent activity of five essential oils against Culex pipiens
. Fitoterapia 2006;77:491-4.
Tripathi AK, Prajapati V, Ahmad A, Aggarwal KK, Khanuja SPS. Piperitenone oxide as toxic, repellent, and reproduction retardant toward malarial vector Anopheles stephensi
(Diptera: Anophelinae). J Med Entomol 2004;41:691-8.
Lamiri A, Lhaloui S, Benjilali B, Berrada M. Insecticidal effects of essential oils against Hessian fly, Mayetiola destructor (Say). Field Crop Res 2001;71:9-15.
Arouiee H. Control of greenhouse whitefly (Trialeurodes vaporariorum
) by thyme and peppermint. KMITL Sc J 2005;5:511-4.
Koschier EH, Sedy KA, Novak J. Influence of plant volatiles on feeding damage caused by the onion thrips Thrips tabaci
. Crop Prot 2002;21:419-25.
Shaiq Ali M, Saleem M, Ahmad W, Parvez M, Yamdagni R. A chlorinated monoterpene ketone, acylated I-sitosterol glycosides and a flavanone glycoside from Mentha longifolia
(Lamiaceae). Phytochemistry 2002;59:889-95.
Joshi RK. Pulegone and menthone chemotypes of Mentha spicata
Linn. from Western Ghats region of North West Karnataka, India. Natl Acad Sci Lett 2013;36:349-52.
Chowdhury JU, Nandi NC, Uddin M, Rahman M. Chemical constituents of essential oils from two types of Spearmint (Mentha spicata
L. and M. cardiaca
L.) introduced in Bangladesh. Bangl J Sci Indus Res 2007;42:79-82.
Bloomquist JR. Chloride channels as tools for developing selective insecticides. Arch Insect Biochem Physiol 2003;54:145-56.
Casida JE, Quistad GB. Golden age of insecticide research: past, present, or future? Annu Rev Entomol 1998;43:1-16.
Chen L, Xue L, Giacomini KM, Casida JE. GABAA receptor open-state conformation determines non-competitive antagonist binding. Toxicol Appl Pharmacol 2011;250:221-8.
Ozoe Y, Niina K, Matsumoto K, Ikeda I, Mochida K, Ogawa C. Actions of cyclic esters, S-esters, and amides of phenyl- and phenylthiophosphonic acids on mammalian and insect GABA-gated chloride channels. Bioorg Med Chem 1998;6:73-83.
Sattelle DB, Lummis SR, Wong JH, Rauh J. Pharmacology of insect GABA receptors. Neurochem Res 1991;16:363-74.
Buckingham SD, Biggin PC, Sattelle BM, Brown LA, Sattelle DB. Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol Pharmacol 2005;68:942-51.
Ozoe Y, Akamatsu M. Non-competitive GABA antagonists: probing the mechanisms of their selectivity for insect versus mammalian receptors. Pest Manag Sci 2001;57:923-31.
Garcia DA, Bujons J, Vale C, Sunol C. Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons. Neuropharmacology 2006;50:25-35.
Garcia DA, Vendrell I, Galofre M, Suñol C. GABA released from cultured cortical neurons influences the modulation of t-[(35)Sbutylbicyclophosphorothionate binding at the GABAA receptor. Effects of thymol. Eur J Pharmacol 2008;600:26-31.
Vale C, Pomes A, Rodriguez-Farre E, Suñol C. Allosteric interactions between gamma-aminobutyric acid, benzodiazepine and picrotoxinin binding sites in primary cultures of cerebellar granule cells. Differential effects induced by gamma- and delta-hexachlorocyclohexane. Eur J Pharmacol 1997;319:343-53.
Hu XJ, Ticku MK. Development pattern of the GABAA-benzodiazepine receptor ionophore complex in primary cultures of cortical neurons. Dev Brain Res 1994;80:137-40.
de Sousa DP, Nobrega FF, de Lima MR, de Almeida RN. Pharmacological activity of (R
)-(+)-pulegone, a chemical constituent of essential oils. Z Naturforsch C 2011;66:353-9.
Tong F, Coats JR. Effects of monoterpenoid insecticides on [3H-TBOB binding in house fly GABA receptor and 36Cl- uptake in American cockroach ventral nerve cord. Pestic Biochem Physiol 2010;98:317-24.
Bessette SM. Pesticidal methods and compositions for modulating GABA receptors. Patents 2010;US 20100144888 A1:US 12/543,406.
Hall AC, Turcotte CM, Betts BA, Yeung WY, Agyeman AS, Burk LA. Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids. Eur J Pharmacol 2004;506:9-16.
de Sousa DP, de Sousa Oliveira F, de Almeida RN. Evaluation of the central activity of hydroxydihydrocarvone. Biol Pharm Bull 2006;29:811-2.
Pizzolitto RP, Herrera JM, Zaio YP, Dambolena JS, Zunino MP, Gallucci MN. Bioactivities of ketones terpenes: antifungal effect on F. verticillioides
and repellents to control insect fungal vector, S. zeamais
. Microorganisms 2015;3:851-65.
Franzios G, Mirotsou M, Hatziapostolou E, Kral J, Scouras ZG, Mavragani-Tsipidou P, Insecticidal and genotoxic activities of mint essential oils. J Agric Food Chem 1997;45:2690-4.
Herrera JM, Zunino MP, Dambolena JS, Pizzolitto RP, Gañan NA, Lucini EI. Terpene ketones as natural insecticides against Sitophilus zeamais
. Ind Crops Prod 2015;70:435-42.
Gordon WP, Forte AJ, McMurtry RJ, Gal J, Nelson SD. Hepatotoxicity and pulmonary toxicity of pennyroyal oil and its constituent terpenes in the mouse. Toxicol Appl Pharmacol 1982;65:413-24.
Thomassen D, Slattery JT, Nelson SD. Contribution of menthofuran to the hepatotoxicity of pulegone: assessment based on matched area under the curve and on matched time course. J Pharmacol Exp Ther 1988;244:825-9.
Moorthy B, Madyastha P, Madyastha KM. Destruction of rat liver microsomal cytochrome P450 in vitro by a monoterpene ketone, pulegone, a hepatotoxin. Indian J Chem Section B 1991;30:138-46.
Madsen CB, Wortzen G, Carstensen J. Short term toxicity study in rats dosed with menthone. Toxicol Lett 1986;32:147-52.
Yerramsetty KM, Rachakonda VK, Neely BJ, Madihally SV, Gasem KAM. Effect of different enhancers on the transdermal permeation of insulin analog. Int J Pharm 2010;398:83-92.
Sanchez-Borzone M, Delgado-Marin L, Garcia DA. Inhibitory effects of carvone isomers on the GABAA receptor in primary cultures of rat cortical neurons. Chirality 2014;26:368-72.
[Figure 1], [Figure 2]