Cytotoxicity and maternal toxicity attributed to exposure to Momordica charantia L. (Cucurbitaceae) dry leaf extract.

Momordica charantia L. (Cucurbitaceae), popularly known as "bitter melon" or "bitter gourd," is a climbing plant well-adapted to tropical countries. This plant is used traditionally to treat several conditions including diabetes mellitus, inflammation, liver dysfunctions, and cancer. Given the widespread ethnopharmacological use, this study aimed to examine the cytogenetic, maternal, and developmental toxicity attributed to exposure to dry extract of M. charantia leaves using Allium cepa and Wistar rats as test models. First, phytochemical characterization of the dry extract by high performance liquid chromatography (HPLC) analyses was performed. Then, Allium cepa roots were exposed to three different concentrations of the dry extract (0.25, 0.5, or 1 mg/ml) to determine the mitotic index, frequency of chromosomal aberrations, and nuclear abnormalities. In addition, pregnant Wistar rats were administered either 500; 1,000 or 2,000 mg/kg dry extract during the gestational period (GD) days 6-15, and subsequently possible toxic effect on the dams and fetuses were recorded. HPLC analyses confirmed rutin as the main secondary metabolite present in the dry extract. In the Allium cepa test, the dry extract was cytotoxic. In Wistar rats, dry extract administration reduced water and feed intake and mean body mass gain, indicating maternal toxicity during the organogenesis period. However, the dry extract did not markedly affect reproductive outcome parameters evaluated. Regarding developmental toxicity assessment, the dry extract treatment did not significantly alter number of skeletal malformations in the offspring. Data demonstrated that the dry extract of M. charantia leaves presents cytotoxicity and low maternal toxicity, indicating indiscriminate use needs to be avoided.

Short and prolonged maternal separation impacts on ethanol-related behaviors in rats: sex and age differences.

Maternal separation (MS) is an animal model widely used to evaluate the influence of early-life stress exposure on ethanol consumption and dependence. The goal of this study was to evaluate the effects of brief and prolonged MS on the pattern of consumption and ethanol conditioned place preference (CPP) in male and female rats during adolescence and adulthood. Wistar rat pups were separated daily from their dams for 15 or 180 minutes during the 2 to 10 postnatal days (PND). In adolescence, half of the litter from each group was evaluated in the ethanol consumption test using the three-bottle test choice paradigm. In addition, using biased procedure, ethanol-conditioned place preference was also evaluated. In adulthood, the other half of the litter was evaluated on the same tests. Our results showed that there are differences in consumption pattern and in alcohol reinforcement between males and females, adolescents and adults. While prolonged MS had no effect on total ethanol consumption in adolescents of both sexes, it induced CPP in these animals. In turn, in adults, previous exposure to prolonged MS increased ethanol consumption without altering ethanol-CPP.Lay summaryGiving the importance of the mother-children (dam-pups when talking about rodents) relationship to proper brain development, the separation of pups from their dam is broadly used as an animal model to study the impact of early-life stress exposure. Here, we used a protocol of brief or prolonged maternal separation to study the impact of early-life stress exposure in the alcohol consumption and conditioned place preference in rats, and how age and sex influence it. We showed that, overall, the prolonged maternal separation increased alcohol consumption in both males and females, but only when animals were tested during the adulthood. In the other hand, prolonged maternal separation increased ethanol conditioned place preference in adolescent rats, both male and female.

[The use of phytotherapy during pregnancy: a global overview]. / O uso da fitoterapia durante a gestação: um panorama global.

The scope of this study is to present an integrative review of the prevalence of the use of phytotherapy during pregnancy. A review of the topic was made by research in the Scielo, Medline and Science Direct databases using the following key words: "herbs and pregnancy," "plant and gestation," with their respective terms in Portuguese. Forty-six articles published between 2000 and 2015 met the study's inclusion and exclusion criteria and were included in this review. Of these, 11 were carried out in Europe, 10 in Asia, 5 in Africa, 3 in Oceania, 16 in America and only one of which was a multinational study. In most of these (67.39%), the interview method was used. A substantial variability in the prevalence of phytotherapy use was reported in the articles. In addition, camomile, ginger, garlic, mint and echinacea were the species most used by pregnant women. Despite the socioeconomic and ethnic-cultural variables among women worldwide, phytotherapy use during gestation is a widespread practice.

Este artigo tem como objetivo realizar uma revisão integrativa da literatura sobre a prevalência do uso da fitoterapia durante a gestação. Foi realizado um levantamento nas bases de dados SciELO, Medline e Science Direct com os descritores "herbal and pregnancy", "plant and gestation" e seus correspondentes em português: "planta e gestação"; "erva e gravidez". Dentre os artigos publicados entre 2000 e 2015, 46 estudos clínicos preencheram os critérios de inclusão e exclusão e foram selecionados para esta revisão. Destes, 11 foram realizados na Europa, 10 na Ásia, 5 na África, 3 na Oceania, 16 na América e, apenas um, foi de caráter multinacional. Na maioria dos estudos (67,39%) o método utilizado foi o de entrevista. A prevalência do uso da fitoterapia descrita nas publicações foi muito variável. Ademais, a camomila, o gengibre, o alho, a menta e a equinácea foram as espécies mais utilizadas pelas gestantes. Os dados mostram que o uso da fitoterapia durante a gestação é uma prática disseminada entre mulheres de todo o mundo, independentemente das variáveis socioeconômicas e étnico-culturais que eventualmente possam distingui-las.

O uso da fitoterapia durante a gestação: um panorama global / The use of phytotherapy during pregnancy: a global overview

Resumo Este artigo tem como objetivo realizar uma revisão integrativa da literatura sobre a prevalência do uso da fitoterapia durante a gestação. Foi realizado um levantamento nas bases de dados SciELO, Medline e Science Direct com os descritores "herbal and pregnancy", "plant and gestation" e seus correspondentes em português: "planta e gestação"; "erva e gravidez". Dentre os artigos publicados entre 2000 e 2015, 46 estudos clínicos preencheram os critérios de inclusão e exclusão e foram selecionados para esta revisão. Destes, 11 foram realizados na Europa, 10 na Ásia, 5 na África, 3 na Oceania, 16 na América e, apenas um, foi de caráter multinacional. Na maioria dos estudos (67,39%) o método utilizado foi o de entrevista. A prevalência do uso da fitoterapia descrita nas publicações foi muito variável. Ademais, a camomila, o gengibre, o alho, a menta e a equinácea foram as espécies mais utilizadas pelas gestantes. Os dados mostram que o uso da fitoterapia durante a gestação é uma prática disseminada entre mulheres de todo o mundo, independentemente das variáveis socioeconômicas e étnico-culturais que eventualmente possam distingui-las.

Abstract The scope of this study is to present an integrative review of the prevalence of the use of phytotherapy during pregnancy. A review of the topic was made by research in the Scielo, Medline and Science Direct databases using the following key words: "herbs and pregnancy," "plant and gestation," with their respective terms in Portuguese. Forty-six articles published between 2000 and 2015 met the study's inclusion and exclusion criteria and were included in this review. Of these, 11 were carried out in Europe, 10 in Asia, 5 in Africa, 3 in Oceania, 16 in America and only one of which was a multinational study. In most of these (67.39%), the interview method was used. A substantial variability in the prevalence of phytotherapy use was reported in the articles. In addition, camomile, ginger, garlic, mint and echinacea were the species most used by pregnant women. Despite the socioeconomic and ethnic-cultural variables among women worldwide, phytotherapy use during gestation is a widespread practice.

Developmental toxicity evaluation of Pimenta pseudocaryophyllus (Gomes) Landrum, (E)-methyl isoeugenol chemotype, in Wistar rats.

BACKGROUND: Pimenta pseudocaryophyllus (Gomes) Landrum (Myrtaceae) has been traditionally used in Brazilian folk medicine. Studies have established the botanical characterization, phytochemistry profile, and pharmacological potential of this species, including antibiotic, anxiolytic, antidepressant, antioxidant, antinociceptive, and anti-inflammatory properties. Despite its widespread use, no previous study has been conducted regarding its toxicological profile, especially during pregnancy. Thus, this study investigated the developmental toxicity of the dry leaf extract of the P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype, in rats. METHODS: First, the dry leaf extract was prepared by a spray-drying technique. Then, pregnant Wistar rats were orally treated with dry extract at doses of 0, 2000, 2500, or 3000 mg/kg from gestational day 6 through 15 (organogenesis period). On gestational day 21, the rats underwent cesarean sections and the reproductive outcomes and biochemistry parameters related to hepatic and renal markers were evaluated. Additionally, the fetuses were examined for external and skeletal variations and malformations. RESULTS: The spray-drying technique preserved the phytocomplex components and showed a satisfactory yield. No relevant differences were seen in the food consumption, reproductive performances, and hepatic and renal biochemical parameters between groups. However, there was a decrease in body weight gain of the dams during the organogenesis period and an increase of minor skeletal variations in the offspring (increased fetal incidences only of delayed ossification of the metacarpals, metatarsals, phalanges, sternebra, and rudimentary ribs) treated with the dry extract. CONCLUSION: The extract of P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype, showed low maternal toxicity and induced minor skeletal variations in the offspring. Birth Defects Research 109:1292-1300, 2017. © 2017 Wiley Periodicals, Inc.

Ethopharmacological evaluation of the rat exposure test: a prey-predator interaction test.

The rat exposure test (RET) is a prey (mouse)-predator (rat) situation that activates brain defensive areas and elicits hormonal and defensive behavior in the mouse. Here, we investigated possible correlations between the spatiotemporal [time spent in protected (home chamber and tunnel) and unprotected (surface) compartments and frequency of entries into the three compartments] and ethological [e.g., duration of protected and unprotected stretched-attend postures (SAP), duration of contact with the rat's compartment] measures (Experiment 1). Secondly, we investigated the effects of systemic treatment with pro- or anti-aversive drugs on the behavior that emerged from the factor analysis (Experiment 2). The effects of chronic (21 days) imipramine and fluoxetine on defensive behavior were also investigated (Experiment 3). Exp. 1 revealed that the time in the protected compartment, protected SAP and rat contacts loaded on factor 1 (defensive behavior), while the total entries and unprotected SAP loaded on factor 2 (locomotor activity). Exp. 2 showed that alprazolam (but not diazepam) selectively changed the defensive factor. Caffeine produced a mild proaversive-like effect, whereas yohimbine only decreased locomotor activity (total entries). Fluoxetine (but not imipramine) produced a weak proaversive-like effect. 5-HT(1A)/5-HT(2) receptor ligands did not change any behavioral measure. In Exp. 3, chronic fluoxetine (but not imipramine) attenuated the defensive behavior factor without changing locomotion. Given that the defensive factor was sensitive to drugs known to attenuate (alprazolam and chronic fluoxetine) and induce (caffeine) panic attack, we suggest the RET as a useful test to assess the effects of panicolytic and panicogenic drugs.

Corticosterone does not change open elevated plus maze-induced antinociception in mice.

It has been demonstrated that the exposure of rodents to the standard elevated plus-maze (sEPM: 2 open and 2 enclosed arms) elicits defensive behavioral reactions and antinociception and also activates the hypothalamo-pituitary-adrenal (HPA) axis. We have recently reported that EPM-induced antinociception is particularly observed when rats and mice are exposed to a totally open EPM (oEPM: 4 open arms). Given that the oEPM seems to be a more aversive situation than the sEPM, we hypothesized that oEPM exposure would induce higher plasma levels of corticosterone than sEPM exposure in mice. In this study, we investigated the influence of exposure to eEPM (enclosed EPM: 4 enclosed arms), sEPM or oEPM on plasma corticosterone levels in mice, with or without prior nociceptive stimulation (2.5% formalin injection into the right hind paw). We also tested whether the nociceptive response in the formalin test and oEPM-induced antinociception are altered by adrenalectomy. Results showed that oEPM-exposed mice spent less time licking the injected paw than sEPM- and eEPM-exposed animals. All three types of EPM exposure increased plasma corticosterone when compared to the basal group, but sEPM- and oEPM-exposed mice showed higher corticosterone levels than eEPM-exposed mice. Prior nociceptive stimulation (formalin injection) did not enhance the plasma corticosterone response induced by the three types of EPM exposure. Indeed, formalin injection appeared to provoke a ceiling effect on plasma corticosterone concentration. Furthermore, neither the nociceptive response in the formalin test nor oEPM-induced antinociception was changed by adrenalectomy. Present results suggest that oEPM antinociception does not depend on corticosterone release in mice.

Ventrolateral periaqueductal gray lesion attenuates nociception but does not change anxiety-like indices or fear-induced antinociception in mice.

The exposure of rodents to an open elevated plus-maze (oEPM: four open arms raised from the floor) elicits naloxone-insensitive antinociception. Midazolam infusion into the dorsal portion of the periaqueductal gray (dPAG), a structure of the descending inhibitory system of pain, failed to alter oEPM-induced antinociception. Chemical lesion of dorsomedial and dorsolateral PAG attenuated defensive behavior in the standard EPM (sEPM), an animal model of anxiety, but failed to change oEPM-induced antinociception. The present study investigated the effects of bilateral lesion, with the injection of NMDA (N-methyl-D-aspartic acid), of the ventrolateral column of PAG (vlPAG) (i) on nociceptive response induced by 2.5% formalin injected into the right hind paw (nociception test) in mice exposed to the enclosed EPM (eEPM: four enclosed arms - a non-aversive situation) or to the oEPM and (ii) on anxiety indices in mice exposed to the sEPM without prior formalin injection. Results showed that oEPM-induced antinociception was not altered by lesion of vlPAG. Nevertheless, the lesion reduced the nociceptive response in mice exposed to the eEPM and increased general locomotor activity during the eEPM and oEPM exposure. Furthermore, vlPAG lesion did not alter anxiety-like indices in mice exposed to the sEPM. The results suggest that vlPAG does not play a role in oEPM-induced antinociception or in defensive reactions assessed in the sEPM. Moreover, vlPAG inactivation induces pain inhibition in mice not exposed to an aversive situation and seems to increase general activity.

Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition

Glutamate N-methyl-D-aspartate (NMDA) receptor activation within the dorsal column of the periaqueductal gray (dPAG) leads to antinociceptive, autonomic, and behavioral responses characterized as the fear reaction. Activation of NMDA receptors in the brain increases nitric oxide (NO) synthesis, and NO has been proposed to be a mediator of the aversive action of glutamate. This paper reviews a series of studies investigating the effects of neuronal NO synthase (nNOS) inhibition in the dPAG of mice in different aversive conditions. nNOS inhibition by infusion of Nω-propyl-L-arginine (NPLA) prevents fear-like reactions (e.g., jumping, running, freezing) induced by NMDA receptor stimulation within the dPAG and produces anti-aversive effects when injected into the same midbrain site in mice confronted with a predator. Interestingly, nNOS inhibition within the dPAG does not change anxiety-like behavior in mice exposed to the elevated plus maze (EPM), but it reverses the effect of an anxiogenic dose of NMDA injected into the same site in animals subjected to the EPM. Altogether, the results support a role for glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice. However, dPAG nitrergic modulation of anxiety-like behavior appears to depend on the magnitude of the aversive stimulus.

Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition

Glutamate N-methyl-D-aspartate (NMDA) receptor activation within the dorsal column of the periaqueductal gray (dPAG) leads to antinociceptive, autonomic, and behavioral responses characterized as the fear reaction. Activation of NMDA receptors in the brain increases nitric oxide (NO) synthesis, and NO has been proposed to be a mediator of the aversive action of glutamate. This paper reviews a series of studies investigating the effects of neuronal NO synthase (nNOS) inhibition in the dPAG of mice in different aversive conditions. nNOS inhibition by infusion of Nù-propyl-L-arginine (NPLA) prevents fear-like reactions (e.g., jumping, running, freezing) induced by NMDA receptor stimulation within the dPAG and produces anti-aversive effects when injected into the same midbrain site in mice confronted with a predator. Interestingly, nNOS inhibition within the dPAG does not change anxiety-like behavior in mice exposed to the elevated plus maze (EPM), but it reverses the effect of an anxiogenic dose of NMDA injected into the same site in animals subjected to the EPM. Altogether, the results support a role for glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice. However, dPAG nitrergic modulation of anxiety-like behavior appears to depend on the magnitude of the aversive stimulus.(AU)

Increased corticosterone levels in mice subjected to the rat exposure test.

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses.

Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis.