Although several studies have shown the inhibitory effects of
Recently, we have shown the efficacy of the hydroethanolic extract obtained from
Few toxicological studies on
The methods used in this study were submitted to and approved by the Institutional Ethics Committee, Federal University of Parana, PR, Brazil, under Protocol number 383, following the international ethics principles for animal experimentation. The experimental design comprises part of the ICH protocol for the analysis of embryonic development, stage B [
Wistar rats were obtained from the Federal University of Parana, Brazil, and maintained under controlled conditions at 22 ± 2°C and a constant 12 h/12 h light/dark cycle. Three-month-old nulliparous rats weighing from 160 to 180 g were used in this study. The male rats were previously mated to attest fertility. The day of sperm detection in the vaginal smear was considered day 0 of pregnancy, and these females were randomly separated into the treatment and control groups and were housed individually in polypropylene cages (414 × 344 × 168 mm). Standard food pellets (Nuvital, Curitiba, PR, Brazil) and tap water were available
The leaves of
The leaves were air-dried in an oven at 40°C for 4 days and then cut and pulverized. The resulting dried powdered plant material was macerated for 7 days in 90% ethanol solution. The solvent was removed using a rotary vacuum evaporator under reduced pressure and lyophilized, giving up 15.39% of the dry material extracted (HETM). The chemical composition of HETM was previously described [
The HETM doses were prepared according to the higher therapeutic dose capable of inducing diuresis and hypotension [
Animals were randomly distributed into four experimental groups of 10–15 animals each: control (aqueous solution 5 mL/kg/day), HETM 3 (
At first, in order to evaluate the HETM toxicity on the maternal organism, the pregnant rats were observed during a 60-minute period after being treated with this substance. This procedure aimed to identify clinical symptoms of toxicity, such as piloerection, behavioral alterations (hyper- or hypoactivity, head flicking), tremors, convulsion, and death. Furthermore, food and water intake were monitored, once a day, and body weight gain or loss was monitored every two days [
The pregnant rats were euthanized by decapitation and autopsied on the 8th day of pregnancy. The liver, kidneys, adrenal glands, uterus, and ovaries were dissected and weighed. Organ weights are reported as absolute and relative weights.
The uterine horns were sectioned longitudinally and the number of implants was determined for the calculation of (1) the implants per group ratio (number of implants/number of corpora lutea) × 100 and (2) the preimplantation losses per group (100 − implantation rate) [
Additionally, blood samples were collected and dehydroepiandrosterone (DHEA), estradiol, and progesterone levels were measured in serum using a microparticle enzyme immunoassay (AxSYM Estradiol and Progesterone assay). Kits were purchased from Abbott Laboratories (Abbott Park, IL, USA).
The data obtained were processed through variance analysis (ANOVA), followed by Bonferroni’s test, in those cases in which samples were normal and homoscedastic. For the data without homoscedastic samples and normal distribution, the nonparametric Kruskal-Wallis test was used, followed by the Mann-Whitney test. Chi-square test was used for the analysis of the preimplantation losses per group rate. Differences were considered significant at a probability level of 5% (
No clinical symptoms for maternal toxicity (head flicking, hyper- or hypoactivity, piloerection, tremors, convulsions, or deaths) were observed in any of the experimental groups. Moreover, the estimated food and water intake did not present significant differences between the control and treated groups (data no shown). Also, there were no significant changes in body weight as well as in the absolute and relative weights of liver, kidneys, adrenals, ovaries, and uterus (Table
Maternal variables after treatment with HETM (3, 30, and 300 mg/kg) or control (aqueous solution) from the 1st to the 7th day of pregnancy.
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Parturient dams | 15 | 11 | 13 | 15 |
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Relative organs weights | ||||
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Values are expressed as mean ± S. E. M. using Kruskal-Wallis test followed by Mann-Whitney test.
The daily treatment with HETM (300 mg/kg) significantly increased the serum DHEA (control: 0.41 ± 0.06; HETM 300: 0.90 ± 0.14 ng/mL;
Serum levels of dehydroepiandrosterone (DHEA) (a), estradiol (b), and progesterone (c) of female Wistar rats treated orally with HETM (3, 30, and 300 mg/kg) or control (aqueous solution) from the 1st to the 7th day of pregnancy. Blood samples were obtained on the 8th day of pregnancy. Each bar represents the mean ± S.E.M. of the group. *The significance levels in comparison to control groups (one-way ANOVA followed by Bonferroni’s test); *
Figure
Percentage of preimplantation losses in relation to the total number of corpora lutea in female Wistar rats treated orally with HETM (3, 30, and 300 mg/kg) or control (aqueous solution) from the 1st to the 7th day of pregnancy. The graph shows the preimplantation losses in uterine horns on the 8th day of pregnancy. Each bar represents the mean ± S.E.M. of the group. *The significance levels in comparison to control group (Chi-square test); **
Historically, women have used herbal drugs in pregnancy to treat pregnancy related illnesses and for their own health and well-being. Herbal drugs are often promoted as “natural” and “safe” alternatives to conventional drugs. These claims may especially appeal to pregnant women who are often concerned about their unborn child’s well-being. Nevertheless, in several countries, herbal drugs are not currently subject to the same regulations as conventional drugs, and hence there is little or no testing of purity, safety, or teratogenicity. Still, recently, there has been great focus on adverse effects of herbal drugs in the medical literature; however, there have been few studies on the use of herbal drugs in pregnancy [
Proper embryo implantation is a critical factor determining the success of a pregnancy, and this process depends on both the embryo and the endometrium, which have to develop in a highly coordinated manner. In a successful implantation, the blastocyst attaches to the endometrial epithelial cells and the trophoblastic cells invade through this layer until they reach the endometrial stroma, thus firmly anchoring the embryo into the uterine wall. At the time of implantation, the endometrium undergoes the process of decidualization and becomes morphologically and functionally distinct from their former state [
Another important aspect to be considered refers to problems with the differentiation of the uterus to the receptive state in response to the ovarian hormones estrogen and progesterone. Normally, the “window” of uterine receptivity lasts for a limited time and estrogen levels within a very narrow range determine the duration of the window of uterine receptivity. Although estrogen at different physiological concentrations can initiate implantation, the window of uterine receptivity remains open for an extended period at lower estrogen levels but rapidly closes at higher levels [
On the other hand, when there is evidence that HETM can induce hypotensive and diuretic effect by inhibiting ACE [
In conclusion, the study showed that the HETM presents compounds responsible for increasing levels of DHEA and estradiol and may induce difficulty of implanting the embryo in early stages of pregnancy. Despite the evidence that the ACE inhibitory activity of HETM may contribute to this effect, this does not exclude that other ways operating in an integrated manner or other molecular mechanisms may be involved in the disturbance. Further, the data reported here collaborates with preclinical investigations of this species and contributes significantly to the safety aspects of using this natural product when trying to get pregnant or during pregnancy.
The authors declare that they have no conflict of interests.
The authors are grateful to CAPES, CNPq, and DEGPP/UNIPAR for financial support.