Effects of Annona squamosa extract on early pregnancy in rats.

Summary

Annona squarnosa Linn., family Annonaceae, is said to show varied medicinal effects, including insecticide, antiovulatory and abortifacient. The purpose of present study was to investigate if A. squamosa seed aqueous extract, in doses higher than that popularly used to provoke abortion, interferes with reproductive performance, and to correlate the ingestion of this extract with possible alterations in rat embryonic implantation. Doses of 300 mg/kg (Treated Group I, n = 17) and 600 mg/kg (Treated Group II, n = 12) body wt. were administered by gavage, during days 1 to 5 of pregnancy (preimplantation period). The control group (n = 13) received water in the same manner, during the same period for comparison with experimental groups. The animals were euthanized on day 10 of pregnancy. Treatment of dams during the preimplantation period showed no signs of toxicity, and no alteration in the corpora lutea, implantations and embryo in terms of development numbers. The percentage of preimplantation and postimplantat ion losses in treated groups I and II did not differ from those of control. Treatment with aqueous extract of A. squamosa seeds caused no morphological change in the endometrium. The absence of morphological alterations in uterine epithelial cells in treated groups I and H permitted a viable embryonic implantation, as verified by the number of embryos in development at day 10 of pregnancy. Thus, A. squamosa seed aqueous extract did not interfere with the reproductive performance of pregnant rats.

Key words: Annona squamosa L., sugar apple, antiimplantation, abortifacient, preimplantadon, medicinal plant, rats, toxicology.

Introduction

The use of natural products as alternative therapy is common in countries with populations that do not have access to scientific medical assistance. In Brazil, this phenomenon is enriched by the indigenous culture (DiStasi et al., 1989). In central and Latin America, a decoction or infusion ingestion prepared with plant extracts is used popularly for fertility control, and for producing temporary sterility, possibly by interfering with embryonic implantation. Several plants have been confirmed as antifertility, abortive, uterine stimulant, estrogenic or cytotoxic agents in animals and humans (Farnsworth et al., 1975).

In the rat, zona pellucida lysis occurs on day 5 of pregnancy and the blastocysts then attach to the uterus (Surani, 1975). Giavini et al., (1984) have revealed that drugs and other common chemicals pass from general circulation into uterine fluid and penetrate readily into the embryo before its implantation. But there is little information about the toxic effects of chemicals or physical agents on mammalian embryos during this period (Moore and Persaud, 2000). The preimplantation period of pregnancy is considered to be an "all-ornone" period, i.e., the period during which maternal exposure to exogenous agents may cause either embryolethality or a normal fetus at delivery (Giavini et al., 1990).

A large number of plants has been used in folk medicine for centuries, including Annona squamosa seed extracts, which have been ingested by Brazilians in attempts to promote abortion. The extract is usually prepared using a handful of fresh seeds of A. squamosa, blended with water and filtered through several layers of fine cloth. Annona squamosa Linn. (Annonaceae) has acquired various regional names: sugar apple (North America); anon (Bolivia, Cuba, Panama); anon de azucar, anon domestico (Colombia); anona blanca (Honduras, Guatemala); ata, fruta do conde, fruta de condessa, frutiera de conde, pinha, araticutitaia or ati (Brazil); saramulla, saramuya (Mexico) (Morton, 1987). This plant is reputed to possess varied medicinal properties, such as insecticide, antiovulatory (Vohora et al., 1975), abortifacient (Mishra et al., 1979) and antitumor activities (Nonfon et al., 1990). Despite the fact that ethnomedical information refers to the use of A. squamosa for the control of reproduction (Farnsworth et al., 197 5), laboratory studies on the toxic effects of A. squamosa appear to be limited to a single study by Queiroz-Neto et al. (1997).

The lack of information about the toxic effects of many plants frequently encourages their use in the treatment of diseases. Among some folk uses of plant extracts, we can identify those uses intended to affect reproduction, including contraception or abortion during early pregnancy. However, several laboratory studies have not confirmed the popular use of these plants (Lemonica and Alvarenga, 1994).

Therefore, the purpose of the present study was to investigate if A. squamosa seed aqueous extract, in doses higher than that used popularly to provoke abortion, interferes with the reproductive performance of rats, and to correlate the ingestion of this extract with possible alterations in embryonic implantation of the rats.

Material and Methods

Extraction of plant material

Annona squamosa L. seeds were authenticated by experts from our Botanical Department, (UNESP -- Botucatu, Sao Paulo, Brazil), where a voucher specimen has been deposited. The material was prepared by grinding 6 g of fresh seeds in a blender with 100 ml of water, similar to the folk-medicine preparation method. The material was filtered through several layers of gauze, and the extract was divided into aliquots, stored and maintained in a freezer until use. A sample was evaporated to determine the total extract content (10 mg/ml).

Animals

After a 10-day acclimatization under standard conditions (21 [+ or -] 4[degrees]C, 12-h light/dark), virgin female Wistar rats, weighing 200 [+ or -] 20 g each, were maintained in a temperature and light-controlled room, with access to water and food ad libitum.

Experimental Procedure

Virgin female Wistar rats were mated with adult Wistar males overnight, and the morning when a sperm-positive vaginal smears were detected was considered to be day 1 of pregnancy. The mated females were divided at random into three groups: one control and two experimental groups which received daily treatment by gavage during the preimplantation period (days 1-5 of pregnancy). The doses used in this experiment were higher than those used popularly, because previous studies carried out in our laboratory showed that the popular dose presented no alteration in reproductive performance (data not published). Control females (n = 13) received water, which was also used to dilute the plant extract. The experimental groups were given doses of 300 mg/kg body wt (Treated Group I, n = 17) and 600 mg/kg body wt (Treated Group II, n = 12) of A. squamosa extract.

The maternal weight gain, food and water intake were measured daily during the experiment to determine maternal toxicity. On day 10, pregnant rats were euthanized under ether anesthesia and subjected to post mortem examination. Uterine horns were removed and the number and distribution of implantation, resorption (embryo or fetal death) and embryos in development were recorded. Corpora lutea were also counted in each ovary. To confirm embryonic implantation sites, the uterine horns were placed in Salewski reactive (Salewski, 1964). The percent embryonic loss before implantation was calculated as: number of corpora lutea -- number of implantations x 100/ number of corpora lutea, and used as a measure of antiimplantational effect. Percent embryonic loss after implantation was calculated as: number of implantations -- number of embryos in development x 100/ number of implantations, and used as a measure of abortifacient effect. Some organs considered essential for embryo survival (kidneys, liver, heart and pancr eas) were removed and weighed.

Samples of uterine horns were removed, fixed in Bouin's solution for 24 h at room temperature, dehydrated and embedded in paraffin. Thick sections of 7 [micro]m each were stained with Hematoxylin-Eosin (HE) for morphological analysis of the uterine epithelium.

Statistical evaluation

The proportion of the number of pregnant animals and the difference in the mean number of implantation sites, embryos in development, corpora lutea and organ weights in the treated versus control groups was compared using Analysis of Variance (ANOVA) (Ostle, 1973). The rates of preimplantation and postimplantation losses, and the resorption index were analyzed by Kruskal-Wallis test (Siegel, 1970). P < 0.05 was selected as the level of statistical significance.

Results

The results obtained with the aqueous extract of Annona squamosa are given in Tables 1 and 2. Treatment with A. squamosa extract did not significantly alter maternal weight gain throughout the early pregnancy (Table 1). Food and water intake was similar and showed no significant difference among animals (data not shown). Weights of the liver, heart, kidneys, spleen and pancreas were similar in all the groups (Table 2).

There were 12.61 [+ or -] 2.33 embryos in development in the control group, 12.70 [+ or -] 2.05 and 12.58 [+ or -] 2.81 in Treated Groups I and II, respectively. The resorption index was 0.61% (1 resorption/165 implants) in the control group, 0.46% (1 resorption/217 implants) and 2.50% (4 resorptions/160 implants) in treated groups I and II, respectively. No significant difference was observed with respect to the resorption index among groups (Table 1).

The reproductive performance of the dams treated before the embryonic implantation period was similar to that of the control group (Table 1). There was no significant difference in the mean number of implantation sites or embryos in development. However, the percent of postimplantation loss showed a tendency, which was not statistically significant, to increase in the group that received plant extract. The rate of preimplantation losses did not present a difference as compared to control (Table 1).

In all the groups, the uterine horns presented the endometrium with a simple columnar epithelium containing secretory cells and cilliated cells. The surface epithelium invaginated into the underlying lamina propria, forming uterine glands (Figure 1).

Discussion

Because the weight gain of mothers during pregnancy was not altered by treatment with an A. squamosa extract and because weight loss is considered to be a good indication of toxicity, it seems that this preparation has little to no toxic effect upon the mothers. Another indication of lack of toxicity was the absence of alterations in organ weights in the treated groups.

It is quite probable that A. squamosa seed extract had no embryolethal effect because the number of resorptions, and rates of preimplantation and postimplantation losses were similar among groups. The same number of embryos in development per mother in all the groups suggests no lethal effect of A. squamosa extract on embryos.

The antiimplantational effect may be due to several factors, such as morphological alterations in the uterine epithelium which interfere with embryonic implantation (Damasceno and Lemonica, 1999). Therefore, we also analyzed the morphological structure of the endometrium after treatment with plant extract. In the present study, morphological changes that could be directly related to antiimplantational effect of the plant extract were not identified. The evidence of the integrity of epithelial cells, in association with the development of uterine glands in groups I and II suggests that the uterus was receptive to embryonic implantation. The number of embryos in development at day 10 of pregnancy confirms our hypothesis.

Although the choice of extract preparation in our paper was not in accordance with classic methodology established by phytochemical study, our aim was to prepare an aqueous A. squamosa seed extract similar to that used by women during early pregnancy.

In this paper, the results showed an absence of abortifacient or antiimplantational activities of A. squamosa seed extract in pregnant rats. The discrepancy between the results obtained and the ethnomedicinal use of A. squamosa seed extract as an abortifacient might be explained in several ways, such as the difference in sensibility existing between the species, the dose used, the treatment period, the method of extract preparation, and botanical differences. According to Lemonica (1996), a comparison of the teratogenic effect of chemical substances between humans and animals revealed that laboratory animals are generally more resistant than humans.

It is well known that drugs and other chemicals pass readily from general circulation into uterine fluid and penetrate the embryo during the preimplantation period. Most teratologists in fact believe that after exposure to exogenous insults during the preimplantation period, the embryo either dies or survives unharmed to term because this gestational phase is considered to be an "all-or-nothing" period (Giavini et al., 1990). In this period, stem cells have the capacity to substitute damaged cells, resulting in a possible embryonic development (Carlson, 1996). It is important to emphasize that a chemical substance, present in the plant extract, may not cause lethal effects in the embryo, but may cause embryo or fetal toxicity. Further investigations might be realized to confirm whether the plant extract possesses toxicological effects in the embryo.

The biological effects produced by a vegetable species may also vary. Botanical identification is insufficient to identify medicinal activity in a plant (Lapa et al., 1999). The use of medicinal plants offers difficulties; first, because botanical divergence among similar species is common; second, because specimens of the same species can have dissimilar biological activity because they may have been collected at different stages of growth or in different localities; and third, because it is complicated to control or the chemical composition of vegetable extracts due to the large number of substances present (Evans, 1996).

A preliminary screening of the petroleum ether extract of seeds of A. squamosa was prepared, and the separation of the active component was performed by column chromatography on silica gel and named squamocin (Fujimoto et al., 1988). The presence of a number of different acetogenins with a characteristic butenolide substituent and tetrahydrofuran or bistetrahydrofuran elements, and which contain hydroxyl group in an a-position has also been established (Nonfon et al., 1990). Therefore, an active lipid-soluble annonin might be the active component responsible for the abortive and antiimplantational activities when given to pregnant rats (Mishra et al., 1979), and is not extracted with water, since the rats of our paper that received an aqueous A. squamosa seed extract did not show any alteration in reproductive performance or endometrial structure. Queiroz-Neto et al. (1997) prepared an A. squamosa seed extract similar to ours, given at a dose of 100 mg/kg body wt. to non-pregnant rats, which showed no toxic e ffect. The same authors also consider that the toxic principles possibly present in the seeds of A. squamosa are not removed in an aqueous extract.

A. squamosa extracts that have a folkloric reputation as abortive, when tested for abortifacient effect in laboratory animals, showed no antiimplantational or abortive activities. We hope to point out that although certain plants have been tested in animals for abortive effects, the negative results presented may or may not be valid. Different animal species can vary in their response to different types of compounds. Furthermore, the antiimplantational study carried out was not designed to test for all types of antifertility effects; that is, by using a too-restricted dosing period (Farnsworth et al., 1975).

Thus, we conclude that treatment with the aqueous extract of A. squamosa seeds at the different doses tested, which were prepared similarly to folk medicine preparations and given from day 1 to day 5 of pregnancy, resulted neither in morphological changes of the endometrium, reflected in the viable embryonic implantation, nor in interference with the reproductive performance of pregnant rats.

Table 1. Reproductive performance of Wistar rats treated with an A.
squamosa seed aqueous extract, by gavage, during the preimplantation
period.

                                     Groups

                                     Control

Mated Rats                           13
Corpora-lutea Number (mean [+ or -]
 SD)                                 12.92 [+ or -] 2.10
Implantation Number (mean [+ or -]
 SD)                                 12.69 [+ or -] 2.14
Embryos in Development Number (mean
 [+ or -] SD)                        12.61 [+ or -] 2.33
Preimplantation Loss (%)              1.78
Postimplantation Loss (%)             0.63
Resorption Index                     1/165
                                     (0.61%)

Maternal Weight Gain (g)
(Mean [+ or -] SD)                   27.07 [+ or -] 10.85

                                     Groups

                                     Treated I

Mated Rats                           17
Corpora-lutea Number (mean [+ or -]
 SD)                                 13.17 [+ or -] 1.55
Implantation Number (mean [+ or -]
 SD)                                 12.76 [+ or -] 2.11
Embryos in Development Number (mean
 [+ or -] SD)                        12.70 [+ or -] 2.05
Preimplantation Loss (%)              3.11
Postimplantation Loss (%)             0.47
Resorption Index                     1/127
                                     (0.46%)

Maternal Weight Gain (g)
(Mean [+ or -] SD)                   26.70 [+ or -] 10.41

                                     Groups

                                     Treated II

Mated Rats                           12
Corpora-lutea Number (mean [+ or -]
 SD)                                 13.50 [+ or -] 1.68
Implantation Number (mean [+ or -]
 SD)                                 13.33 [+ or -] 1.56
Embryos in Development Number (mean
 [+ or -] SD)                        12.58 [+ or -] 2.81
Preimplantation Loss (%)              1.26
Postimplantation Loss (%)             5.62
Resorption Index                     4/160
                                     (2.50%)

Maternal Weight Gain (g)
(Mean [+ or -] SD)                   26.17 [+ or -] 12.79

No significant difference was observed, as compared to the control
group.

Table 2. Maternal organ weight of Wistar rats treated with an A.
squamosa seed aqueous extract, by gavage, during the preimplantation
period.

              Groups

Maternal      Control              Treated I
Organs (g)    (n = 13)             (n = 17)

Liver         11.83 [+ or -] 1.41  11.42 [+ or -] 1.57
Pancreas       0.92 [+ or -] 0.23   0.95 [+ or -] 0.29
Right Kidney   0.97 [+ or -] 0.09   1.02 [+ or -] 0.11
Left Kidney    0.94 [+ or -] 0.08   0.99 [+ or -] 0.10
Heart          0.94 [+ or -] 0.09   0.90 [+ or -] 0.10
Spleen         0.66 [+ or -] 0.12   0.74 [+ or -] 0.12

              Groups

Maternal      Treated II
Organs (g)    (n = 12)

Liver         11.76 [+ or -] 1.46
Pancreas       0.89 [+ or -] 0.23
Right Kidney   0.99 [+ or -] 0.11
Left Kidney    0.98 [+ or -] 0.12
Heart          0.90 [+ or -] 0.09
Spleen         0.63 [+ or -] 0.12

Data are reported as means [+ or -] SD. No significant differences was
observed, as compared to the control group.

Acknowledgments

The authors would like to thank Mr. Jose Alexandre do Prado, Experimental Laboratory of Obstetrics, for technical assistance and Mr. Gelson Rodrigues, Anatomy Department, for histological slide preparation

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D. C. Damasceno (1, 2), G. T. Volpato (1), T. C. F. Sartori (3), P. F. Rodrigues (2), E. A. Perin (2), I. M. P. Calderon (1), M. V. C. Rudge (1)

(1.) Experimental Laboratory of Obstetrics, Department of Gynecology and Obstetrics of School of Human Medicine of Botucatu -- UNESP -- Sao Paulo State, Brazil

(2.) Discipline of Human Embryology of School of Human Medicine of Marilia (FAMEMA), Silo Paulo State, Brazil

(3.) Disciplie of Human Histology of School of Human Medicine of Marilia (FAMEMA), Silo Paulo State, Brazil

Address

Profa. Dra. Debora C. Damasceno, Disciplina de Embriologia Humana, Faculdade de Medicina de Marilia (FAMEMA), Av. Monte Carmelo, 800. CEP. 17519-030, Marilia, Sao Paulo, Brasil.

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