Journal of Ethnopharmacology 43 (1994) 39-44

Journal of ETHNO-

PHARMACO[DG~

Abortive and teratogenic effect of Acanthospermum hispidum DC. and Cajanus cajan (L.) Millps. in pregnant rats

I.P. Lemonica*, C.M.D. Alvarenga

Departamento de Farmaeologia, Laborat6rio de Teratologia Experimental, UNESP, Campus de Botucatu, 18618-000 Botucatu SP, Brazil

Received 19 May 1993; revision received 23 December 1993; accepted 24 March 1994

AImraet

Extracts of Acanthospermum hispidum and Cajanus cajan have been used by Brazilian people in an attempt to pro- duce abortion. In order to evaluate the possible abortive and/or teratogenic effect of these plant extracts, female Wistar rats were treated with the aqueous extract (infusion, proportion C. cajan and A. hispidum 1:1.3). Doses of 0, 150, 300 and 600 mg/kg were daily administered by gavage during the organogenic period. The animals were sacrificed at term. There was no significant change in the mean weight of the fetuses, and rto change in the percentage of post implantation loss in the treated groups. However, there was an increase in the number of external malformations, and this was re- lated to dose. No internal malformations were observed in fetuses at term, but there was a significant incidence of fetuses with visceral anomalies. The tendency of the pregnancy to continue or terminate did not change with the treatment.

Keywords: Embriotoxicity; Abortive plant effects; Teratogenicity

1. Introduction

The use of natural principles as an alternative therapy in countries where a large percentage of its population does not have access to medical assis- tance is widespread. In Brazil this fact is enriched by a cultural tradition inherited from the Indian culture (Di Stasi et al., 1989). The plant extracts are traditionally used by the major part of the needy population living mainly in the countryside or by the population who leave the countryside to

* Corresponding author.

live in the city. The lack of information about the toxic effects of many of these plants frequently in- duces their use in the cure of diseases. Among some folk uses of plant extracts we can identify those uses intended to affect reproduction, such as contraceptive or abortive agents during early preg- nancy. However, laboratory studies do not con- firm the popular use of these plants.

Cajanus cajan (L.) Millps. (Leguminosae) has many popular uses: the extract from the leaves, for example, has been claimed to promote elimination of little renal calculus (Corr~a, 1926). Seeds of Acanthospermum hispidum DC. have toxic effects

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40 I.P. Lemonica, CM.D. Alvarenga / J. Ethnopharmacol. 43 (1994) 39-44

characterized by hemorrhage, weakness and diar- rhea (Ali and Adam, 1978). These authors showed that ingestion of the bud or seed of Compositae is toxic to mice, causing lesions in the kidneys, liver, heart, lungs, intestines and bilear vesicle.

Literature data show the presence of ses- quiterpenoids and phenolic compounds in A. hispidum and that other species of this family pre- sent cytotoxic and anti cancer activity (Di Stasi et al., 1989). The plants of Compositae and Legnminosae families are popularly used for their abortive, oxytoxic or anti fertility properties (Farnsworth et al., 1975; Camargo, 1985).

It has been reported that extracts of two species of these plants, used together, have been ingested by Brazilians in an attempt to promote abortion. The extract is usually prepared using a handful of fresh leaves of C. cajan and the same amount of the fresh whole plant (stalk, seeds, leaves and roots) ofA. hispidum, put in a container with 1000 ml of boiling water. The container is covered until it reaches room temperature and the infusion is then drunk after filtration.

Our purpose is to evaluate experimentally, in rats, the relationship between the ingestion of extracts in doses commonly used to provoke abor- tion of A. hispidum and C. cajan and the occur- rence of malformations, anomalies or fetal deaths when this extract is ingested during the organo- genic period.

hispidum and C cajan, respectively). The infusion was prepared by pouring 100 ml of boiling water on a flask containing 10 mg of the powdered plants. The flask was kept under agitation and covered until reaching room temperature. After filtration the dried weight was determined (40 mg/ml), and the infusion divided in aliquots, kept at -20°C. The lowest dose used in this experiment corresponds to that used as an abortive by hu- mans. Control females were treated with saline, which was also used to dilute the plant extracts.

The females were weighed on days 1, 7, 14 and 21 of pregnancy, when they were sacrificed. After the removal of the uterine horns, the number of corpora lutei, implants, resorptions, live and dead fetuses were recorded.

The fetuses were weighed and examined for macroscopic external malformations. Half of each litter was fixed in Bouin's solution for subsequent visceral examination according to Wilson's meth- od (1965), and the other half was stained with Alizarin red according to the technique of Staples and Schneli (1964) to reveal alterations of the skel- eton. The degree of ossification was evaluated using the parameters proposed by Aliverti et al. (1979).

Data were analysed by Goodman's test (1964, 1965), the Krushal-Wallis test (Siegel, 1975) or analysis of variance (Ostle, 1973).

2. Materials and methods 3. Results

After 10-day acclimatization in standard condi- tions (21 ± 4°C, light from 06:00 h to 18:00 h), virgin female Wistar rats originally weighing 200 ± 10 g, were mated. The day on which sper- matozoa were found in the vaginal smear was defined as day 1 of pregnancy.

The mated females were randomly assigned to different experimental groups and treated during the organogenic period. The treatment consisted of the ingestion by gavage of 150, 300, or 600 mg/kg/day of 10% aqueous extracts of A. hispidum (leaves, stalk and seeds) and C cajan (leaves). These parts of both plants were dried at 40°C in an aerate stove, ground and their powder used to prepare an infusion at the ratio of 1.3:1 (A.

The reproductive performance of the dams, presented in Table 1, showed that the treatment did not cause any change in the resorption num- ber, the same occurring with the number of live fetuses, and post implantation loss. The maternal

• weight gain was lower in all treated groups; how- ever, these data do not have statistical significance, presenting parallelism and coincidence in the tested periods.

There was a dose-related tendency for the inci- dence of external malformations to increase (Table 2), but without statistical significance. The percen- tage of affected litters increased in all groups that received the extract, but no dose-related effect was evident. However, the group that received 300

LP. Lemonica, C.M.D. ,41varenga / J. EthnopharmacoL 43 (1994) 39-44 41

Table 1 Effect of different doses of aqueous extract from A. hispidum and C cajan mixed together given by gavage, during the organogenic period, on reproductive performance of pregnant Wistar rats

Group I Group II Group 11I Group IV Control 150 mg/kg 300 mg/kg 600 mg/kg

Number of females Mated 12 Pregnant at term 12 With total resorption 0

Maternal weight gain (mean -4- S.D.; g) 106.0 -4- 24.2 No. of corpora lutea (mean -4- S.D.) 13.6 -4- 2.2 No. of implantations (mean -4- S.D.) 12.4 -4- 2.5 No. of live fetuses (mean -4- S.D.) 12.0 -4- 2.5 Resorptions 5 Postimplantation loss 3.2% Fetal weight (mean -4- S.D.; g) 4.1 -4- 0.4 Placental weight (mean -4- S.D.; g) 0.5 -4- 0.1

12 12 12 12 11 12 0 1 0

82.9 ± 16.6 82.0 ± 40.8 86.0 ± 32.9 13.4 ± 2.7 13.1 ± 2.1 12.3 ± 2.1 !1.2 ± 2.0 9.7 ± 5.0 10.3 ± 3.7 10.7 ± 1.8 9.3 -4- 4.8 10.2 -4- 3.6 5 4 1 3.8% 3.8% 0.8% 4.0 -4- 0.3 4.1 -4- 0.4 4.1 -4- 0.4 0.5 .4- 0.I 0.5 -4- 0.I 0.5 -4- 0.I

mg/kg/day, showed the highest percentage of af- fected litters (41.7%).

The visceral analyses, the data of which are presented in Table 3, showed that the treatment did not lead to the occurrence of visceral malfor- mations. On the other hand, the animals of the groups that received doses of 150 and 600 mg/kg/day, presented an increased number of fetuses with anomalies, the urinary system being the most frequently affected.

Skeletal malformations were not found in the experimental groups (Table 4). Skeletal anomalies,

although found, were present, both in the control and in the treated groups.

The fetal and placental weights were similar in the experimental groups, as were the number of ossification centers, shown in Table 4. This shows that the fetuses of all experimental groups presented the same degree of development.

4. Discussion and conclusions

Many plants of the Compositae and Legumin- osae families have a folk use as an abortion

Table 2 Effect of different doses of aqueous extract from A. hispidum and C cajan mixed together given by gavage during organogenic period of pregnant Wistar rats on the incidence of external malformations of the fetuses

External malformations Group I Group II Group Ill Group IV control 150 mg/kg 300 mg/kg 600 mg/kg

Affected fetuses 1/144 (0.69%) Affected litters 1/12 (8.33%) Palatoschisis 1 Subcutaneous edema Acampsia Acromicria Ear heterotopic Cranial alterations Micrognathia Gastroschisis

4/129 (3.10%) 4/12 (33.33%)

5/I 12 (4.46%) 7/122 (5.74%0) 5/12 (41.67%) 3/12 (25%) 2 5

2 2

I 1 1 1

42 L P. Lemonica, C M.D. Alvarenga / J. Ethnopharmacoi. 43 (1994) 39-44

Table 3 Effect of different doses of aqueous extract from A. hispitfion and C. cajan mixed together given by garage during orgenogenic period of Wistar pregnant rats on the incidence of visceral malformations and anomalies of the fetuses

Group I Group II Group III Group IV Control 150 mg/kg 300 mg/kg 600 mg/kg

Visceral malformations Affected fetuses 0/70 1/60 0/54 0/57 Affected litters 0/11 1/12 0/10 0/11 Hydrocephalia - - 01 - - - - Anophthalmia - - 01 - - - -

Visceral anomalies Affected fetuses 16/70 28/60* 17/54 28/57* Affected litters 08/11 11/12 08/10 I 1/11 Enlarged and/or sinuous ureter 13 20 15 22 Ectopic testis 03 03 01 0 Ectopic kidney 03 04 02 06

*P < 0.05, significantly different from controls.

p romote r (Camargo, 1985), but jus t a few of them had this abor t ive p roper ty exper imental ly con-

f irmed (Farnswor th et al., 1975). The aqueous extract f rom both A. hispidum and

C. cajan mixed together given to pregnant rats th roughout the organogenic per iod did not cause abor t ion . This effect might occur before implanta- tion, which would conf i rm the abor t ive folk use of these plants. The number o f resorpt ions and per- centage o f post implanta t ion loss was similar in all

groups. This indicates tha t the doses used d id not p roduce an embriole thal effect in rats. On the o ther hand, the da ta on reproduct ive per formance showed that the number o f p regnant females was the same at the beginning and at the end o f the experiments, except in the group that received 300 mg/kg where there was a female that d id not coa- t inue the pregnancy. This fact has no statist ical

significance, and was not repeated when the doses were increased.

Table 4 Effect of different doses &aqueous extract from A. hispidum and C cajan mixed together given by gavage during organogenic period of pregnant Wistar rats on the incidence of skeletal malformations and anomalies of the fetuses

Group 1 Group il Group II1 Group IV Control 150 ms/kg 300 mg/kg 600 mg/kg

Skeletal malformation Affected fetuses 0•62 0•67 0/58 0•59 Affected litters 0/10 0/12 0/10 0/I I

Skeletal anomalies Affected fetuses 37/62 35/67 33/58 30/59 Affected litters 10/10 11/12 10/10 i i/11 Sternal anomalies 28 25 21 21 Vertebral anomalies 14 6 5 6 14th ribs 12 9 8 3 Number of ossification centers 17.52.4- 1.11 18.52 4- 0.90 17.67 4- 1.07 18.45 4- 1.07

(mean 4- S.D.)

I.P. Lemonica, C.M.D. AIvarenga /J. Ethnopharmacol. 43 (1994) 39-44 43

During the experimental period, the behavior of the dams was observed and their food and water ingestion and body weight gain were recorded to establish if the treatment caused toxicity.

Some authors reported the use of plant extracts causes teratogenicity in animals. However, this effect occurred only when the administered doses were toxic to the dams (Keeler and Crowe, 1984; Seno et al., 1984; Panter et al, 1985). As our goal is to observe if there is a correlation between inges- tion of the plant extract by women for the purpose of causing abortion and the occurrence of malfor- mations in newborns, we used doses that did not present toxicity to the dams. The presence of toxic- ity in the mothers can itself cause an increase in malformations (Khera, 1984); therefore the real effect of the extract could not be observed.

The doses of the employed plant extracts varied from a minimum dose, which corresponds to the daily ingestion by pregnant women, to a maximum dose that could not cause toxicity in the dams. This corresponded to an initial dose of 150 mg/kg and a final dose 4 times that quantity.

The analyses of the occurrence of malformation showed that 25-41% of the litters treated with the plant extracts presented external malformation and that the number of affected fetuses rose from 0.69 to 5.7 in these litters. This demonstrated a biological tendency of the ingestion of the plant extract to cause an increase in the number of exter- nal malformations.

The analyses of the anomalies showed that there was an increase in the number of visceral anomal- ies in two groups that received the plant extracts, not dose related. The skeletal anomalies consisted prevalently of extra ribs, external and vertebral anomalies; however, they are frequently found in our strain of rats.

On the other hand, the weight of the examined fetuses was similar in all experimental groups. This was also the case with the number of ossification centers, and showed that a similar degree of de- velopment occurred in both the treated and con- trol groups.

In short, it can be said that the extracts of these plants used in increasing doses, after embryo im- plantation, did not modify the reproductive per- formance of female pregnant rats. The present

data give no indication of embriotoxicity, although the extract had been administered during the organogenic period. Neither do the data give any indication in relation to the abortive effect. The effects observed with, and probably caused by, this treatment were an increase in the number of fetal visceral anomalies and a tendency to increase the occurrence of external malformations. The results suggest that the possible abortive effect of the extract of these two plants used together may occur in the pre-implantation period (all or none of the period of the pregnancy).

Acknowledgments

We wish to thank Dr C.R. Padovani for statistical analyses and Mr P.C. Mioni for techni- cal assistance.

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