Abstract
Context: Mentha x villosa Hudson (Lamiaceae) is an aromatic herb employed as a food spice. In folk medicine, it leaves are used as a tranquilizer and anti-hypertensive, even by pregnant women.
Objective: There are no reports about its effects in gestation and exposed fetuses, the aim of this study.
Materials and methods: At gestation day (GD) 01, 24 rats were divided in four groups: one control and three experimental groups (n = 6/group). The experimental groups received, by gavage, from GD06 to GD16, 10, 25 or 50 µg/kg/day of Mentha x villosa essential oil. The control group received the vehicle (Tween 80 and distilled water − 2%). The parameters of body weight gain, water and food intake were recorded. At GD20 the females were euthanized. Half of the fetuses from each litter were directed for the study of visceral malformations and the remaining fetuses for the study of skeletal malformations.
Results: The statistical analyses revealed absence of alterations in body weight gain, water and food intake, litter weight, fetuses number and weight, reabsorptions and implantations. The treatment revealed absence of visceral and skeletal malformations. The visceral analysis revealed mild hemorrhagic points at brain, but more numerous at kidney, liver and blood vessels near heart, in some fetuses from some experimental litters.
Conclusion: The essential oil was not able to promote impairment to the pregnant rats and to gestation. Even occurring lack of malformations, fetotoxicity was revealed by mild hemorrhagic points at liver, kidney, brain and blood vessels of some exposed fetuses.
Keywords::
Introduction
Medicinal plants exert pharmacological actions in the body, promoting curative proprieties and preventing a range of afflictions. They are often employed by populations without professional advice under the impression that they are harmless, even when used chronically or during gestation, justifying the importance of studies directed to the elucidation of the toxicity and risk of teratogenic effects.
Mentha sp. species are employed as aromatic, for cooking because of their flavoring properties, and as medicinal plants, usually as teas and infusions. The numerous species are a result from cross pollination, yielding new hybrids. Actually, these species are employed as gums, tooth paste and by pharmaceutical industries. The leaves contain vitamins A, B and C and minerals such as calcium, phosphorus, iron and potassium (CitationBraga, 1983). The leaves promote tonic effects, stimulate the digestive system and present antiseptic, antinociceptive and anti-parasitic proprieties (CitationLeal-Cardoso & Fonteles, 1999).
Mentha x villosa Hudson (Lamiaceae) is an aromatic herb, known in Brazilian folk medicine as ‘small leaved mint’, ‘creeping mint’ or ‘regular mint’ (CitationBraga, 1983; CitationAlmeida, 1993; CitationLahlou et al., 2001). This species, known also as Mentha crispa, is a hybrid from the crossing between Mentha spicata L. and Mentha suaveolens Ehrh. The plant has many pharmacological activities: anti-parasitic, tranquilizer (central nervous system depressant), bradicardizant, anti-hypertensive, analgesic, relax smooth musculature and contract skeletal musculature (CitationAlmeida et al., 1996; CitationFogaça et al., 1997; CitationLahlou et al., 2001; Citation2002a,ba,Citationb; CitationSousa et al., 2009). An extract obtained from leaves present santispasmodic effect and is employed for the treatment of intestinal disorders caused by Giardia lamblia and Entamoeba histolytica (CitationSantana et al., 1992; CitationSousa et al., 1997). Previous phytochemical analyses revealed the compounds 1,2-epoxipulegone-rotundifolone (main constituent), carvone, menthol, linalol, cineol, cetone, mentone, d-carvone, reduced quantities of mentonone, pipertinone, jasmone, acetic esters and mentone isovaleric, flavonoids, rosmarinic acid, luteoline, tannic acid and resine as constituents of it leaves. From the essential oil obtained from the leaves were detected 28 compounds: 13 monoterpenoids, 10 sesquiterpenoids, one phenylpropanoid, one ester and three others not identified (CitationMatos et al., 1999; CitationMartins et al., 2007).
Despite the wide range of use of Mentha x villosa in folk medicine, there are few references in literature about its effects in fertility and no information about effects at gestation and exposed fetuses. The only study found in reproduction was developed with an alcoholic extract. It revealed that the extract was not able to promote fertility impairment and a contraceptive effect in male rats (CitationDimech et al., 2006). Considering that pregnant women can employ this species as a medicine (tea or essential oil) and as a food spice, the aim of the present study was to elucidate if Mentha x villosa essential oil impairs gestation and whether it is toxic to fetuses exposed during gestation.
Material and methods
Essential oil
Fresh leaves of Mentha x villosa were collected at Itacuruba city (Pernambuco-Brazil) at October 2009. Its botanical identity was determined by Dr. Francisco J. Abreu Matos (Laboratório de Produtos Naturais, UFC). A voucher specimen (#27286) is deposited in the Prisco Bezerra Herbarium (UFC). The essential oil was obtained using well established methodology (CitationLahlou et al., 2002a) at the Chemistry Laboratory of Natural Products, Department of Chemistry, Universidade Federal da Paraíba (Brazil). The extraction was done by Dr. Marçal de Queiroz Paulo, who kindly provided the essential oil for use in this study. Mentha x villosa fresh leaves (10 kg) were subjected to steam distillation for 8 h. The oil obtained (0.1%) in the usual way (CitationCraveiro et al., 1976; CitationGuedes et al., 2004a), was dried over sodium sulfate and stored at 4°C.
Animals
In this study, 24 adult Wistar rats (250–270 g) about 90 days old were used. The rats, from the same strain, were obtained from the vivarium of the Health Sciences Center at the Universidade Federal do Rio Grande do Norte. Two female rats were placed together with one male in the afternoon. The next morning, females showing evidence of mating (vaginal smear with sperm = gestation day 1) were housed in plastic cages (n = 1/cage) measuring 40 × 50 × 20 cm, under controlled temperature (22 ± 2°C), with a 12:12 h light:dark schedule and had free access to food and water. The animals used in this study were maintained in accordance with the Ethical Principles in Animal Research adopted by National Ethic Research Committee (CONEP/MS) and approved by the Ethical Committee of Animal Use (CEUA) from Universidade Federal do Rio Grande do Norte (protocol 041/2010).
Treatment
Pregnant Wistar rats (n = 24) were divided at gestation day (GD) 01 (the day on which sperm were found in the vaginal smears of females in estrus) into four groups (n = 6/group), one control and three experimental groups. The experimental animals received from GD06 to GD16 (organogenesis period), by gavage, 10, 25 or 50 µg/kg/day of the essential oil prepared in 2% Tween 80 (20 µL Tween 80 for 1 mL distilled water) solution. The control group received the vehicle, by gavage at the same period. During the gestation period the parameters of body weight, body weight gain, water, and food intake were recorded. At GD20 the pregnant rats were euthanized with thiopental sodium (70 mg/kg; i.p.). After exteriorization of the uterine horns, the parameters litter weight, number of fetuses, number of dead fetuses, body weight and body length of the fetuses per litter, macroscopic physical alterations, number of implantation sites and reabsorptions in each uterine horn, number of corpora lutea in each ovary, were measured. The pre- and post-implantation losses (%), implantation (%) and live fetuses (%) ratios were calculated. The organ weight/body weight ratios (liver, kidney, spleen and pancreas) of the pregnant rats were calculated. To investigate the teratogenic action of the essential oil, half of the fetuses from each litter were directed for the study of visceral malformations, developed employing methodology described by CitationWilson (1965). The last half fetuses of each litter were directed for the study of skeletal malformations employing methodology described by CitationTaylor (1986). The fetal observations were recorded in accordance with the internationally developed terminology (CitationWise et al., 1997; CitationPaumgartten et al., 2009).
Statistical analysis
The data were analyzed using the analysis of variance ANOVA and Tukey Kramer post hoc test when necessary. In all cases, results were considered significant when p < 0.05.
Results
The essential oil was analyzed by CG/MS at the Chemistry Laboratory of Natural Products from Universidade Federal da Paraíba as described previously (CitationLahlou et al., 2002a). The following constituents were found: α-pinene, β-pinene, 1,8-cineol, limonene, trans-ocimene, linalool and the majority compound rotundifolone (piperitenone oxide) − 35.4%, corroborating with previous studies (CitationFogaça et al., 1997; CitationGuedes et al., 2002; CitationMartins et al., 2007).
The statistical analysis (ANOVA) revealed that the essential oil, at the doses employed, was not able to promote toxicity to the pregnant rats due to the absence of alterations in body weight, body weight gain (), water and food intakes () and organ weight/body weight ratios (data not shown) of the dams. The essential oil, at the doses used, was not able to promote impairment at gestation of the treated pregnant rats, investigated by recording some parameters as litter weight, number of fetuses, ovaries weight () and by calculating the pre and post-implantation, implantation and live fetuses ratios (). The visceral and skeletal analyses revealed absence of malformations in experimentally exposed fetuses ( and ). However, the visceral analysis revealed mild hemorrhagic points in the brain (2 fetuses from group 10 and 1 fetuses from group 50), liver (8 fetuses from group 10, 7 fetuses from group 25 and 4 fetuses from group 50), kidneys (6 fetuses from group 10, 7 fetuses from group 25 and 4 fetuses from group 50) and blood vessels near heart (4 fetuses from group 10, 7 fetuses from group 25 and 2 fetuses from group 50).
Table 1. Parameters recorded to evaluate gestation impairment of pregnant rats treated, during organogenesis, with Mentha x villosa essential oil.
Table 2. External examination data of litters of rats exposed, during organogenesis, to Mentha x villosa essential oil.
Table 3. Visceral examination data of litters of rats exposed, during organogenesis, to Mentha x villosa essential oil.
Table 4. Skeletal examination data of litters of rats exposed, during organogenesis, to Mentha x villosa essential oil.
Figure 1. Food and water intake of pregnant rats treated, during the organogenesis period, with Mentha x villosa essential oil.
Figure 2. Body weight and body weight gain of pregnant rats treated, during the organogenesis period, with Mentha x villosa essential oil.
Discussion
In folk medicine, the leaves of M. x villosa are employed as an infusion, decoction or by soaking the leaves (CitationCartaxo et al., 2010). Generally, a person eats or drinks an infusion obtained from 100 g of fresh leaves daily. This corresponds to a dose of 1.7 g of leaves/kg body weight. Considering the mean body weight for humans about 60 kg, and that 1 kg of fresh leaves yields 1 mg of the essential oil (CitationCraveiro et al., 1976; CitationGuedes et al., 2004a,Citationb), the dose ingested daily by a human being to treat diseases, in folk medicine, is approximately 1.7 µg/kg. In our study, developed with rats, we employed the 10, 25 and 50 µg/kg doses. These doses were adopted considering the animal species and other ways of human exposure, as in the form of local phytotherapeutic medicines (CitationGuedes et al., 2004b) and as food spice. In addition, considering some previous studies, developed with the essential oil or aqueous extracts obtained from fresh leaves, to elucidate pharmacological actions in rodents, the doses adopted are inside the range of doses employed before (CitationGuedes et al., 2004a,Citationb).
Our essential oil, as previously reported (CitationFogaça et al., 1997; CitationLahlou et al., 2001; CitationGuedes et al., 2004; CitationMartins et al., 2007; CitationSousa et al., 2009), presents monoterpenoids, sesquiterpenoids and, as a major active compound, rotundifolone (data not shown).
It is well defined by the scientific community that alterations in body weight, food and water intake, are the first signs that indicates toxicity (CitationArchibong & Abdelgadir, 2004; CitationBarlow et al., 2002). In our study, alterations at body weight (), body weight gain, food and water intakes and organ weight/body weight ratios were not observed in the experimental dams, suggesting that the essential oil, at the doses and treatment period adopted, was not able to promote toxicity to these animals.
Evaluation of fertility and gestation result in important information about the consequences of exposure of chemical agents in the reproductive system (US EPA, Citation1996). A study developed with an alcoholic extract obtained from leaves of Mentha x villosa showed that male rats pre-treated with 0.5 and 1.0 g/kg of the extract for 30 days did not show fertility impairment nor promoted alterations at gestation and reproductive performance of no treated dams mated with these males after treatment ending period (CitationDimech et al., 2006). In another study, an aqueous extract from M. arvensis promoted fertility impairment, decreased number and motility of sperm and reduced testis and epididymis size and weight in mice treated for 20 days (CitationSharma & Jacob 1996, Citation1997, Citation2001, Citation2002).
The main objective of our study was to investigate the teratogenic potential of Mentha x villosa essential oil. For this reason, the experimental protocol adopted was different from the studies described above (CitationSharma & Jacob 1996, Citation1997, Citation2001, Citation2002). We treated the pregnant rats during organogenesis. Our study revealed absence of toxicity and no alterations at gestation of the treated pregnant rats, as demonstrated in . The pre- and post-implantation losses, live fetuses and implantation ratios were not different between treated and control dams. These results suggest that Mentha x villosa essential oil administered to pregnant rats during the gestation period at the doses employed was not able to promote reproductive alterations.
It is known that the embryo exposed to teratogens during pre- and post-implantation fatally leads to embryo lethality. At organogenesis, this exposure can promote malformations (CitationJelinek, 2005). At the fetal period, functional or structural anomalies can occur in fetuses as a result of the exposure to teratogens. Those anomalies generally occur at the central nervous, reproductive or immune systems and may cause to the exposed person/animal, from child to adult age, behavioral and/or reproductive disturbances and/or auto immune diseases (CitationHoyer, 2001; CitationJelinek, 2005). The visceral and skeletal analyses performed (, and ) revealed the absence of malformations at fetuses exposed to the essential oil during organogenesis period. However, mild hemorrhagic points at the brain, and more numerous at the liver, kidneys and blood vessels near the heart were found in a few fetuses of a few litters exposed to the three doses of the essential oil, as in .
It was previously reported that an aqueous extract (CitationGuedes et al., 2004a,Citationb) and the essential oil (CitationLahlou et al., 2001; CitationMartins et al., 2007), both obtained from fresh leaves of Mentha x villosa, present rotundifolone, an oxygenated monoterpene (cetone), also known as piperitenone oxide or 1,2-epoxipulegone, as major constituent. A vasorelaxant action due to endothelium dependent (via EDRfs, at least NO and prostacyclin) and endothelium independent (calcium channel blockade) mechanisms was attributed to rotundifolone (CitationGuedes at al., 2004a). Hypotention, as a consequence of the essential oil direct cardiopressant action and peripheral vasodilatation, was observed in adult rats (CitationLahlou et al., 2001). The way this compound acts during organogenesis at heart and blood vessels formation may be able to promote the mild hemorrhagic points observed in a few fetuses of some experimental litters. It is well known that humans and animals treated during organogenesis with statins can give birth to abnormal fetuses. The statins are vasorelaxant medicines classified by FDA as category X in pregnancy (not major teratogens). It is employed in the treatment of preeclampsia but the treatment of artherosclerosis, by inhibition of cholesterol synthesis, has been stopped during pregnancy because of the risk to fetuses (CitationEdison & Muenke, 2004; CitationKazmin et al., 2007; CitationCostantine et al., 2010). Considering that Mentha x villosa also present vasorelaxant action, we speculate the hemorrhagic points found in liver, kidney, brain and vessels near heart as a consequence of this effect during organogenesis. However, more studies need to be developed to confirm this hypothesis even because the hemorrhagic events occurred in a few fetuses of a few litters treated with the different doses of the essential oil.
In Brazil, the use of Mentha x villosa essential oil is not restricted to folk medicine only. It is used as spice and as active constituent of local phytotherapeutic medicines. In addition, the leaves of this plant are frequently employed in folk medicine, even by pregnant women, as teas, infusions and syrups made with honey. Considering the numerous ways the plant and essential oil are used by humans and the lack of information about it effects at gestation and exposed fetuses, our data provides some knowledge about the exposure to Mentha x villosa essential oil, during organogenesis critical period.
Conclusions
Our study suggests that Mentha x villosa essential oil is not toxic to pregnant rats and does not impair gestation at the doses employed when administered during organogenesis period. However, even with the absence of skeletal and visceral malformation observed in the exposed fetuses, this study revealed that the essential oil presents fetal toxic effect, due to the hemorrhagic points viewed at the brain, but more frequently, at the kidney, liver and vessels near heart of some fetuses exposed to the three doses (10, 25 and 50 µg/kg) employed during organogenesis. More studies need to be performed, with a larger number of litters, to clearly establish this fetal toxic effect of Mentha x villosa essential oil.
Acknowledgments
The animals, food and standardized place for animals were kindly provided by the Central Biotherium from Centro de Ciências da Saúde – UFRN. We thank UFRN for making possible this study. Special thanks are to the pharmacy students Cássio Oliveira Mourão, Mylena Mylana Freire da Cunha and Alisson Eduardo Florêncio by helping with the statistical analyses, tables and figures elaboration.
Declaration of interest
The authors declare no conflicts of interest.
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