Teratogenic Evaluation of 80% Ethanol Extract of Embelia schimperi Vatke Fruits on Rat Embryo and Fetuses

Introduction. Embelia schimperi Vatke (family Myrsinaceae) is a commonly consumed anthelminthic plant in Ethiopia. The plant has significant efficacy in treating intestinal worms. However, there are limited data about the safety/toxicity of the plant. Moreover, the teratogenic effect of the plant is not yet well studied despite significant number of Ethiopian mothers consuming herbal medication during their pregnancy. Purpose. This study aimed to evaluate the teratogenic effect of the hydroalcoholic extract of E. schimperi fruit on rat embryos and fetuses. Methods. Pregnant albino Wistar rats were treated with 80% hydroalcoholic fruit extract of E. schimperi at 250 mg/kg, 500 mg/kg, and 1000 mg/kg dosage, whilst the controls were pair-fed and ad libitum groups. Maternal food intake, maternal weight gain, number of implantations, number of prior resorptions, fetal viability, fetal weight, fetal and embryonic crown-ramp length, placental weight, placental gross morphology and histopathology of placental tissue, number of somites, embryonic system, gross/visceral morphological malformations, and ossification centers were evaluated as teratogenicity indices. Results. The crude extract of E. schimperi did not exhibit a significant difference in most developmental indices including the development of a circulatory system, nervous system, and musculoskeletal systems among treated animals and the controls. However, histopathological evaluation of placentas from the treatment groups showed that inflammatory reactions and calcifications compared to the pair-fed and ad libitum controls. Conclusion. Administration of the 80% hydroalcoholic extract of E. schimperi fruit during the period of organogenesis in rats did not show a significant toxic effect on embryonic and fetal developmental indices. However, it might affect the structural integrity of the placenta as it is evidenced by inflammatory reactions and calcifications of decidua basalis of rat placenta. Fresh fruits of E. schimperi were collected from Debre Markos localities located 305 km northwest of the capital Addis Ababa, Ethiopia. The plant was identified and authenticated by a botanist at the Department of Plant Biology and Biodiversity Management, Addis Ababa University, where a voucher specimen (collection number ZA001) was deposited for future reference. Fruits of E. schimperi were dried at room temperature for two weeks at the herbarium of Traditional and Modern Medicine Research Directorate (TMMRD) of Ethiopian Public Health Institute (EPHI). The dried fruits were then ground using an electronic grinding mill. The powder was first defatted using n-hexane in a Soxhlet apparatus. This was followed by maceration of 100 g of the marc 1 L of 80% ethanol in a ratio of 1 : 10 (w/v). As a result, a yield of 12.8% dried crude extract was obtained. Nulliparous female albino Wistar rats weighing 220–250 g, which were not subjected to the previous experimental procedures, were used. The animals were maintained in a stainless-steel metallic cage at room temperature (22 ± 3°C) with a relative humidity of 50%–60% under a controlled alternating 12-hour light-dark cycle. Animals were acclimatized for 5 days prior to the experiment. During the period of adaptation, all the animals received food (pellet) and tap water ad libitum. Factors thought to bring fetal losses which were not treatment-related such as unnecessary handling of pregnant animals and stress from external factors like noise was minimized [12]. Mating was carried out by introducing a male albino Wistar rat of proven fertility into a cage containing two virgin female rats. Day-1 of gestation was determined the next morning after microscopic examination of vaginal smear to look for the presence of sperm cells. For those female rats with no sperm cells, a vaginal smear was repeated and examined the next morning. The male rat was maintained inside the same cage until confirmation of pregnancy [13, 14]. Pregnant rats were grouped randomly into five groups containing 10 pregnant rats in each group. The first three groups were treated with 250 mg/kg (Group I), 500 mg/kg (Group II), and 1000 mg/kg (Group III) of the crude extract suspended in distilled water, (1 ml/100 gm of body weight) [12] with free access to tap water and food. The animals in Group IV were categorized as a pair-fed control group and were supplied with the mean daily food intake of the previous three groups of animals with distilled water. The pair-fed control group was intended to evaluate if there would be a difference in the outcome variables due to food intake variation in the previous three groups. Group V animals were labeled as ad libitum control group taking food and water without restriction. The ad libitum group was designed to evaluate the effect of animal handling during administration of the crude extract. The doses were calculated based on previous studies [7, 10]. The extracts were administered through oral gavage. Experiments were carried out on 12 days old rat embryos and 20 days old fetuses. During both experiments, the treatment period was from day-6 through day-12 of gestation. The rationale behind selecting this period for treatment was due to the fact that this period represents a period of active embryogenesis and organogenesis. As a result, it was designated as the critical periods of rat development. Cage side clinical observation of animals was done once daily for possible signs of behavioral and physical changes throughout the experimental period. Coma, convulsions and tremors, eyes, feces consistency, fur and skin, mortality, mucous membrane, respiration, salivation, somatomotor activity, behavior pattern, and urination (colour) were the parameters during cage side evaluation as signs of toxicity. Cage side evaluation was done every 30 minutes for the first 4 hours after administration of the crude extract [15]. In order to avoid bias generally, we used double blinding manner during handling of lab animals, administration of the crude extract, and evaluation of the outcome variables. Each of the aforementioned activities were carried out anonymously by different individuals who had no clue about the grouping of animals and the given treatments. The purpose of this experiment was to evaluate the teratogenic effect of E. schimperi on 12 days old rat embryo. At the end of the treatment period (day-12 of gestation), the dams were anesthetized by injecting sodium pentobarbital 150 mg/kg intraperitoneally [16, 17]. Laparotomy was done to reveal the gravid uterine horns which was later dissected along the antimesometrial border to divulge the developing embryos. With the aid of GXM-XTL3T101 dissecting stereomicroscope and fine forceps, the membranes were removed along with the adjacent maternal tissue to reveal the embryo surrounded by a yolk sac. At this juncture, the yolk sac circulation became clearly visible and was evaluated thoroughly. The yolk sac was then removed to evaluate embryonic developmental indices like the embryonic nervous system, sensory organs, and musculoskeletal systems. These variables were examined according to the Brown and Fabro morphological scoring system [18] (Supplemental file) which was adopted for in vivo teratogenicity studies by Belete et al. [19] and Abebe et al. [16]. These experiments were carried out in 20 days old rat fetuses. The goal was to evaluate the potential toxicity of E. schimperi on fetomaternal outcomes and fetal developmental indices in near-term rat fetus. The weight of each pregnant animal was recorded on 1st, 6th, 12th, and 20th day of gestation. Food intake for every 24 hours was weighed the next morning at a constant time starting from day-1 of gestation up to day of sacrifice. Similarly, administration of the plant extract was done daily at a constant time [20]. On the day of sacrifice (day-20 of gestation), the dams were anesthetized by injecting sodium pentobarbital. Laparotomy was done to reveal the gravid uterus (Figure 1). Gravid uteruses were explanted immediately after the euthanasia and placed in a broad Petri dish. A careful incision was made along the antimesometrial border of the uterus guided by a dissecting microscope (GXM-XTL3T101 stereo microscope). The fetuses were revealed by removing the fetal membranes and detaching them from their respective placentas. After revealing the gravid uterus, the number of implantation sites and prior resorptions was counted and recorded. Alive/dead fetuses were counted after applying gentle pressure on them. Once the fetal membranes and other maternal tissues were removed, the fetuses were weighed using a calibrated digital balance (Mettler AE160). Crown-ramp length (CRL) was measured for every fetus. Placental weight was also recorded before histopathological tissue processing. 12 days old embryos from rats treated with 1000 mg/kg crude extract of E. schimperi. (a): Embryo enclosed by its yolk sac (YS) with visible vitelline vessels (VV), distinguishable head (HD), and tail (T) regions. (b): CNP (cranial neuropores/closed), FL (fore limb), HL (hind limb), ME (mesencephalon), PA (pharyngeal apparatus), RE (rhombencephalon), S (somite), and TE (telencephalon). Three placenta sample tissues were taken from each animal for gross examination and further histopathological analysis. Samples were initially fixed with 10% formaldehyde for 24 h. Then, tissue processing was carried out using an automatic tissue processor (Leica, TP 1020, Germany). The steps were arranged to start with dehydration by ascending gradient of alcohol concentration followed by clearing and impregnation by xylene and melted paraffin wax, respectively. The tissues were then embedded in paraffin wax and ready for sectioning. The thickness of the tissue to be sectioned by the microtome was adjusted to 5 μm for light microscopy. Finally, the tissues were stained by a hematoxylin and eosin technique [16, 21]. Later, the histological slides were examined by a senior pathologist under a light microscope for indices of functional as well as structural changes in the placenta [22, 23]. Fetuses were revealed by removing the fetal membranes and detaching them from their respective placentas. Afterward, each fetus was fully examined for the presence of gross structural malformations of craniofacial development, limb development, vertebral column, tail development, and external genitalia. After fetuses were fixed in Bouin’s solution (picric acid 75%, formalin 25%, and glacial acetic acid 5%), visceral/soft tissue evaluation was conducted by a free-hand razor blade sectioning technique based on a recommendation by Seegmiller et al. as a modified Willison’s technique [16]. The legs and tail were removed at the place of their attachment to the trunk before making a transverse cut between the jaws by a sharp blade. This will help to evaluate the palate for any cleft. Subsequently, coronal slices were made through the head to evaluate the presence of hydrocephalus, ventricular enlargement of the brain, and nasal septum defect [16]. Further transverse sections were made along the trunk to evaluate the possible existence of cardiovascular, respiratory, and abdominal defects. This experiment was designed to study the effect of E. schimperi on the process of bone formation on 20 days old rat fetus after staining the bones of the rat fetus. Three fetus/litter were sampled and further processed based on the Rigueur and Lyons method [24]. The initial step was euthanizing the fetus with pentobarbital followed by tissue permeabilization and skin removal facilitated by bathing for 30 sec in 60°C hot water. Afterwards, evisceration was done by making an abdominal incision. The eviscerated samples were immersed in a solution containing a fixative solution, 90% ethanol, for 24 h. The samples were then transferred to a container filled with 1% potassium hydroxide (KOH) for a purpose of soft tissue removal. Subsequently, the samples were stained for 24 h by a solution of alizarin red (0.005%) at 4°C to obtain an optimum level of skeletal staining. For those samples presumed to be over stained by alizarin red, Mall’s solution (79% distilled water, 20% glycerin, and 1% KOH) was used as a correction chemical. Finally, each sample was stored in an increasing concentration gradient of glycerol till examination. Hyoid bone, sternum, ribs, vertebrae, and bones, the upper and lower limbs were assessed against Nash and Persaud’s skeletal scoring chart [25]. Data were entered and analyzed using the Statistical Package for Social Science (SPSS) software version 24. The statistical results were exhibited in terms of the mean (μ) and the standard deviation (SD). One-way analysis of variance (ANOVA) with the post Hoc (Turkey) test and Chi-square test at P < 0.05 level of significance was employed to look over significant statistical differences among experimental groups. Results of placental histopathology were presented qualitatively based on predefined parameters [19, 26]. Ethical approval letter (Ref no. AAUMF03-008) was obtained from the Institutional Review Board (IRB) of the College of Health Sciences, Addis Ababa University with a protocol number 021/19/Anat in biddableness with OECD test guideline (TG-414/2018) [12]. Experimental animals were humanly handled based on the guidelines for ethical conduct in the care and use of nonhuman animals in research by American Psychological Association (APA) [27]. TMMRD/EPHI laboratory standards were also strictly followed in humanely disposing sacrificed rats.