Abstract: The tea made from Luffa acutangula fruits has been largely used by women for abortion induction and abortions occurred in ruminants that had ingested the fruits. The research aimed to study the abortive effect of L. acutangula tea in rats. Eleven pregnant Wistar female rats were used. On the 15th gestational day, six rats were dosed with 10 mL kg^-1 of L. acutangula tea (50 g of dried fruit in 100 mL of water) and the other five rats were dosed with saline solution. On the 21st gestational day, all the rats were submitted to cesarean section. There was no difference between the two groups on body weight and body weight gain and no sign of maternal toxicity was observed in females throughout the experiment. The number of points of implantation, live and dead fetuses, corpora lutea and points of reabsorption did not differ between groups but it was found reduced fetal weight in the group treated with L. acutangula. The search for external malformations revealed a fetus with cleft palate from a plant-treated mother. No lesion was found at histological evaluation of placentas. In conclusion, the ingestion of L. acutangula during pregnancy may promote developmental toxicity.

INTRODUCTION

Plants of the Cucurbitaceae family are characteristically but not exclusively, producers of cucurbitacins, highly oxygenated triterpenes. Cucurbitacins isolated from plants of the genus Luffa have various pharmacological properties including anti-inflammatory, antimicrobial, antitumoral and hepatocurative effects and they are cytotoxic (Miro, 1995). Other compounds isolated from Luffa species include luffaculins, which are ribosome-inactivating proteins (Ng et al., 1992; Chan et al., 1994; Lin et al., 2002; Wang and Ng, 2002; Hou et al,. 2007), the trypsin inhibitors LA-1 and LA-2 (Haldar et al., 1996) and a lectin that is specific for chito-oligosaccharides (Anantharam et al., 1986).

The tea made from fruits of Luffa acutangula fruits has been widely used by women for induction of abortion and as a purgative (Lorenzi and Matos, 2002). Furthermore, several farmers from the northeastern region of Brazil have reported abortions in ruminants that had ingested fruits of this plant (Da Silva et al., 2006). However, studies of the interference of this plant with reproduction are needed. The research aimed to study the abortifacient effect of L. acutangula tea in rats.

MATERIALS AND METHODS

Mature fruits of L. acutangula Roxb. were collected at Catole do Rocha municipality, Paraíba state, Brazil. Botanical identification was done by Prof. Odaci Fernandes de Olivera, a retired professor from Universidade Federal Rural do Semi-Árido (UFERSA), Mossoró, RN, Brazil. A total of 50 g of dried powdered fruits were added to 100 mL of distilled water. The mixture was warmed until it began boiling and it was then filtered. The solution obtained was kept refrigerated for up to 7 days.

Adult male and virgin female Wistar rats were obtained at the age of about 12 weeks from the Department of Animal Sciences, Universidade Federal Rural do Semi-Árido, Mossoró, RN, Brazil. Food and tap water were provided ad libitum. For mating, two females were placed together with one male. Females that showed evidence of mating (a vaginal plug, or a vaginal smear showing sperm cells) were assigned in rotation to each dose group until the required 12 females had been allotted to each group. The day on which there was evidence of mating was recorded as day 0 of gestation. During gestation, the female rats were housed individually in plastic cages measuring 40x50x20 cm, which were covered with metal lids. The animal room was maintained at 22-24°C and on a 12-h light/dark cycle. Eleven pregnant Wistar female rats were used for the experiment. On the 15th day of gestation, six rats were dosed by gavage with 10 mL kg^-1 of the infusion of L. acutangula and the other five rats were dosed with 10 mL kg^-1 of saline solution. On the 21st day of gestation, all the rats were submitted to Cesarean section.

The number of points of implantation, live and dead fetuses, corpora lutea and reabsorption points was recorded. Fetuses and placentas were weighed individually and the fetuses were evaluated carefully for the presence of external malformations. The placentas were collected for pathological study. Paraffin-embedded sections were stained with Hematoxylin and Eosin (H and E).

Data are reported as the mean±standard deviation and they were compared using the unpaired t-test for parametric data or the Mann-Whitney test for non-parametric data using GraphPad Prism v. 4 for Mac. The level of significance was set at p<0.05.

RESULTS

The body weight and gains in body weight of rats dosed with L. acutangula did not show significant (p>0.05) differences from control rats (Table 1). Furthermore, no clinical signs of maternal toxicity were observed.

The data on reproduction are presented in Table 2. The number of points of implantation, live and dead fetuses, corpora lutea and points of reabsorption did not differ (p>0.05) between groups but reduced (p<0.05) fetal weight was found in the group treated with L. acutangula.

Table 1:	Body weight and gain in body weight (in g) of pregnant rats dosed with Luffa acutangula or saline solution (control) on the 15th day of pregnancy. The data are presented as means followed by the respective standard deviation

Table 2:	Reproductive performance (mean±SD) of pregnant rats dosed with Luffa acutangula or saline solution (control) on the 15th day of pregnancy
p<0.05 (unpaired t test)

Fig. 1:	Heads from fetuses of pregnant rats dosed with Luffa acutangula (above) or saline solution (below) at 15 days of pregnancy. Note presence of cleft palate in fetus from treated rat

The search for external malformations revealed a fetus with a cleft palate from a female rat treated with L. acutangula (Fig. 1). The histological evaluation of the placentas showed no lesions on treated and control rats.

DISCUSSION

Poisonous plants often affect animal reproduction, causing dysfunction including abortions, infertility and teratogenesis. In Brazil, poisonous plants that promote abortions and neonatal loss include Aspidosperma pyrifolium Mart. (De Souza Lima and Soto-Blanco, 2010), Ateleia glazioviana Baill. (Stolf et al., 1994), Tetrapterys acutifolia Cav., Tetrapterys multiglandulosa A. Juss. (Tokarnia et al., 1989, 1998) and Stryphnodendron obovatum Benth. Several other species occasionally promote abortions, for example Amaranthus spinosus L. (Peixoto et al., 2003) and cyanogenic plants (Soto-Blanco and Gorniak, 2004).

The treatment with L. acutangula did not affect maternal body weight and no clinical signs of maternal toxicity were observed. Thus, no evident toxicity was produced in dams. It is well known that maternal toxicity is responsible for disturbances of fetal development because maternalfetal interactions are affected (Khera, 1984; Chernoff et al., 1989). In the study, any possible interference in fetal development from maternal toxicity may be considered improbable. In the study, the fetal weight was reduced in the group treated with L. acutangula but other reproductive data did not show significant variation. The reduced fetal weight is a significant effect because this weight is one of the most important parameters measured in studies of developmental toxicity (Christian, 2001). Thus, it can be stated that L. acutangula presents a fetotoxic effect.

Luffa acutangula is popularly used by women who aim to interrupt gestation (Lorenzi and Matos, 2002). Furthermore, several farmers from the northeastern region of Brazil have reported abortions in ruminants that had ingested fruits of L. acutangula (Da Silva et al., 2006). Several studies have identified proteins from L. acutangula and other Luffa species that have abortifacient action (Schilling and Heiser, 1981; Ng et al., 1992; Chan et al., 1994; Lin et al., 2002; Wang and Ng, 2002) including the proteins luffin b and lufaculin (Chan et al., 1994). These proteins promote the inactivation of ribosomes (Ng et al., 1992; Chan et al., 1994; Wang and Ng, 2002; Hou et al., 2007), which affects the synthesis of cellular proteins and consequently, embryogenesis and fetal development. In the study, the post-implantation loss in rats dosed with L. acutangula was 21.5%, which is much higher than that of the control group (5%) but this result did not reach statistical significance.

The search for external malformations revealed a fetus with a cleft palate from a female rat treated with L. acutangula. To the knowledge, this is the first time that a malformation has been attributed to L. acutangula. However, it has been reported that Luffa operculata was responsible for significant alterations in the palatal epithelium of the frog (Menon-Miyake et al., 2005). Several chemicals are known to induce cleft palate, including the non-steroidal anti-inflammatory drug acetylsalicylic acid (DePass and Weaver, 1982), the corticosteroid dexamethasone (Bonner, 1984), the solvent xylene (Hood and Ottley, 1985), the anticonvulsant sodium valproate (Alsdorf and Wyszynski, 2005) and the environmental contaminant dioxin (Bock and Köhle, 2006).

The placenta is an important organ that performs a number of different and specialized functions in pregnancy. Several chemicals may promote damage to the placenta and the damaged placenta may affect embryonic and fetal development (Goodman et al., 1982; Soto-Blanco et al., 2009). In the research, no lesions were found on histological evaluation of the placentas of treated rats. Thus, the toxic effects of L. acutangula on the fetuses could not be secondary to a damaged placenta.

CONCLUSION

The ingestion of L. acutangula during pregnancy may promote developmental toxicity. In rats, this was shown by reduced fetal weight and the occurrence of cleft palate.