The Effect of Aqueous Extract of Cecropia glazioui Snethlage ( Embauba ) in the Rat Fetal Development

The effect of aqueous extract of Cecropia glazioui Snethlage (Embauba) in the rat fetal development. This study was to complement previous assays on the physical and neurobehavioral development of rats resulted from oral administration of 1 g/kg/day Cecropia glazioui Snethlage (C. glazioui) aqueous extract (LD50 > 5 g·kg) in pregnant rats (periand post-natal studies). In the present study, the effect of 2.5 g/kg/week C. glazioui aqueous extract, administered to pregnant rats during 15 days, was verified in the rat offspring development. No acute or chronic toxicity (no effects on mortality or weight average daily gain) were observed. In addition, no effects on reproductive parameters (offspring vitality, placenta and fetus weight, number of corpora lutea on each ovary, preand post-implantation loss) and on offspring external morphology were found. We concluded that C. glazioui aqueous extract administered during pregnancy did not cause abnormalities in rat offspring.


Introduction
Several studies concerning the pharmacological effects of Cecropia glazioui Snethlage (Cecropiaceae), popularly known as "Embauba" in Brazil, were published in the last decade.The C. glazioui aqueous extract showed anxiolytic-like effect in mice 1, probable by blocking monoamines uptake in the central nervous system 2.An anti-asthmatic property was confirmed in guinea pigs treated with C. glazioui purified fraction 3.It also showed hypotensive activity 4, which is not related to angiotensin-converter enzyme 5, and it has antisecretory/antiulcer properties 6.Plants are an important source of new molecules, which result mainly as consequence of their ontogenesis caused by internal and external forces 7.They produce active primary and secondary metabolites to defend themselves against predators, microorganisms, and UV rays; and to attract seeddispersive animals and insects 8.Some of C. glazioui constituents, such as catechins, procyanidins and flavonoids, were observed by Tanae et al. 9.Brazilian legal requirements of phytotherapics' marketing demand data on their pharmacological action and therapeutic efficacy.Safety is other legal aspect regarding phytotherapics 10-15.However, few studies on phytotherapic safety are available in the literature and data regarding C. glazioui toxicity is still scarce.A previous study showed that the daily exposure to 1 g/kg C. glazioui aqueous extract showed low toxicity to pregnant rats and their litters 16.The objective of the present study was to evaluate the influence of weekly oral administration of C. glazioui aqueous extract (2.5 g/kg) in pregnant rats, simulating the human sporadic use.

Plants
The Cecropia glazioui Snethlage specimen were collected in Tapirai city (State of São Paulo, Brazil).A voucher specimen is deposited in University of Sorocaba (UNISO) herbarium, being identified by Dr Sérgio Romaniuc Neto (Botanic Institute of São Paulo, Brazil).

Preparation of the Aqueous Extract
Fresh leaves (450 g) of C. glazioui without petiole were dried, powdered and a 70% hydroalcoholic extract was obtained by percolation.The extract was concentrated under reduced pressure and lyophilized providing 102.3 g of powder (efficiency = 22.7%).It was stored at room temperature without light and humidity until the toxicological assays were performed.The C. glazioui aqueous extract was freshly prepared in distillated/deionizated water before oral administration.

Animals
Wistar rats (160 -200 g) of both genders were obtained and kept at UNISO/Pharmacy School facilities according to "The Guide for the Care and Use of Laboratory Animal" (National Research Council 1996) and "European Community Guidelines" (EEC Directive of 1986; 86/609/EEC).All animals were maintained in groups (5 -6 rats/cage) with food and water ad libitum.A twelvehour light/dark cycle at constant temperature (23˚C ± 1˚C) was observed.All animals were previously adapted to laboratory conditions during one week before the experiments.The study design was approved by the UNISO Ethical Committee for Experiments.

Reproductive Ability Evaluation
Twelve sexually-naive rat females were mated with rat males (five females and one male per cage).Pregnancy was confirmed through the presence of spermatozoids in vaginal-washing rubbing observed by microscopy analysis 17.The presence of spermatozoids was considered as the first day of pregnancy.Pregnant females were kept in separate cages.At the 1 st , 5 th , 10 th and 15 th days of pregnancy, six females received 2.5 mL/kg of deionized water by gavage (control group) and six females received 2.5 g/kg of C. glazioui solution by gavage (treated group).According to Gerenutti et al. 16, this C. glazioui dose is non-toxic for adult female rats.The posology of C. glazioui was based on rat-fetus development (in days from ovulation): blastocyst formation-3 to 5 days; implantation-5 to 6 days; organogenesis-7 to 17 days 18.The weight gain of the pregnant rats exposed to any chemical agent during a specific period is one of the most used parameters to determine toxicological effects 19,20.At the 18 th day of pregnancy, each rat female was anesthetized by halothane (Halotano ® , Cristalia, Brazil) inhalation and the uterus was rapidly excised.The following macroscopic parameters were evaluated in order to observe the reproductive performance 20,21: 1) Offspring vitality; 2) Placenta and fetus weight (grams); 3) The number of corpora lutea on each ovary; 4) Pre-implantation loss (%) = corpora lutea numberimplantation number/corpora lutea number 5) Post-implantation loss (%) = implantation numberalive fetus number/implantation number Afterward, offspring animals were killed by halothane inhalation, fixed in Bouin's solution for 24 -48 h, and kept in 70% hydroalcoholic solution in order to measure the following parameters (in cm): antero-posterior (A) and latero-lateral (B) of cranio; antero-posterior (C) and latero-lateral (D) of thorax; cranio-caudal (E) and tail (F), as showed in Figure 1.

Statistical Analysis
The data were analyzed by using Student's t test.The significance level was set at 5%.The results were expressed as mean ± standard error mean (SEM).

Results
Figure 2 shows the body weight (in grams) gain during pregnancy and killing period (18 th day).At the 1 st day of pregnancy, the females of C. glazioui group showed lower (p < 0.05) body weight gain when compared to control group.There was no influence (p > 0.05) of C. glazioui in body weight in the further periods.No other changes, such as morbidity or mortality, were registered during the experimental period.
Figure 3 shows the weight of placenta and fetus.No statistically significant difference (p > 0.05) was observed between control and treated groups for both parameters.
The Table 1 shows the reproductive performance of pregnant rats exposed to C. glazioui (n = 6) compared to the control group (n = 6).C. glazioui did not influence (p >    Offspring vitality (%) 100 100 0.05) any of the five reproductive performance parameters.
Figure 4 shows the measurement (in cm) of offspring external anatomical parameters.No statistically significant difference between C. glazioui and control groups was observed regarding any of the measurements.require developmental and reproductive toxicology (DART) tests for drugs destined for human use.DART studies require at least one of three segments of reproductive cycle 18: 1) premating and mating through implantation (reproduction and fertility studies); 2) from implantation through major organogenesis (teratology and/or development toxicological studies); 3) late pregnancy and post-natal development (the perinatal/postnatal studies).In the present study, the segment 2 was observed, while segment 3 was previously studied by Gerenutti et al. 16.Those authors observed the effect of C. glazioui extract (Cg) on physical and neurobehavioral development of rats.Female rats received 1 g/kg/day of Cg during pregnancy and they observed that LD 50 was higher than 5 g/kg.In addition, latency of uprightness and negative geotaxis reflexes were enhanced by Cg in comparison to the control group, but the rearing frequency decreased 18.They concluded that Cg showed low toxicity to the pregnant rats and their litters, and these effects were similar to the anxiolytic-like effect observed in mice by Rocha et al. 1.In our study, the second segment of reproductive cycle, commonly referred as the major period of organogenesis (6 to 15 days in rats), was focused in a new experimental design.This included the period from implantation to closure of the hard palate, being the Cg extract administered at critical points (1 st , 5 th , 10 th and 15 th days of pregnancy).No signs of morbidity, mortality or abortion were registered during the pregnancy period, and no evidence of chronic toxicity, food intake changes, or body weight gain (except at the 1 st day) was induced by Cg, which is similar to the results observed by Gerenutti et al. 16.Abnormalities are usually classified as malformations or developmental variations.Malformations are structural anomalies altering general body conformity, causing dis-ruption or interfering with body function, being generally incompatible with life.Developmental variations are defined as anatomical structure alterations having no significant biological effect on health or body conformity, usually representing slight deviations from normal 22.Considering the offspring external morphological measurements in the present study, Cg extract did not induce significant effects in these measurements when compared with the control group.Studies carried out to establish the potential ability of drugs to induce adverse effects on the fetal development, which used acceptable and rationale experimental design, could provide accurate extrapolation of the potential risk in human beings of that particular drug.Considering the previous study performed by Gerenutti et al. 16, which used the same aqueous extract of Cg, it was possible to conclude that Cecropia glazioui Snethlage presents no toxicity on morphological development of rat offspring.

Figure 3 .
Figure 3. Mean (±SEM) of weight gain of placenta and fetus.No statistically significant changes were observed between groups (p > 0.05).