Introduction

From time immemorial, extracts from the leaves, seeds, fruits, barks and roots of plants have been used in the preparation of syrups and infusions in traditional medicine. These preparations have been used to treat cases ranging from the common cold to malarial, liver chirrosis, hypertension, and so on. The active constituents contributing to these protective effects are the phytochemicals, vitamins and minerals (Okwu & Ekeke; 2003). Extracts from the roots, barks seeds and fruits of these plants are used in the preparation of syrups and infusions in traditional medicine for the treatment of various ailments. One of such plant is Aspilia africana (compositae). It is a semi-woody herb from a perennial woody root stock up to 2m high. The plant has served as food sources for cattle, sheep, hare and rabbits. Its phytochemical analysis reveals that it is rich in saponins, crude oil protein, sterols and terpenes.

Aspilia africana is one of the plants that contain a wide range of biological activity, including antiviral, fungicide and antibacterial due to the presence of thiarubrines, a derivative of 1,2-dithiocyclohexa-3,5-diene (Masato & Wu, 1994). Those plants that contain these rare compounds are used in popular medicine of equatorial Africa as remedy for abdominal infections. Review has it that leaves of Aspilia africana are swallowed wholly by chimpanzees to get rid of endoparasites (Rodriguez & Wrangham, 1992). It is believed to have an anti-parasitic compound extracted in the intestine of the animal without necessarily digesting the leaves.

The cold extract of Aspilia africana appears to have a broad spectrum antibacterial activity because both Gram-positive and Gram-negative bacteria were sensitive to the extracts (Adeniyi & Odufowora, 2000).

Tamura et al (1997) reported that OSW-1 (3 beta, 16 beta, 17 alpha-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)- (1-->3)-(2-O-acetyl-alpha-L-arabinopyranoside)), a steroidal saponin, decrease levels of ovarian steroids due to its direct inhibitory action on the gene expression of the steroidal enzyme and on the proliferation of granulosa cells in the ovary. Studies on contraceptives of plant origin are important because the synthetic hormonal contraceptives in current use produce some side effects. Records on folk medicine mention 225 species of plants with contraceptive and abortive properties. However, experiments performed on animals have confirmed the contraceptive effect in only 145 of these plant species. Chemically, these drugs belong to a wide variety of classes including terpenoids, alkaloids, glycosides, phenols, and other compounds (Korkhov & Mats, 1983). These chemicals are richly present in Aspilia africana.

It is the aim of present work to find out the effects of methanolic extract of the leaves of Aspilia africana on the histo-architecture, weights of the ovaries and general body weights of the rats as indices for its reproductive toxicity.

Material And Methods

Animals

25 cyclic female rats were sorted randomly selected from the animal house of the Igbinedion University, Okada, Edo State. The rats were fed on standard rat pellet produced by Bendel Feed and Flour mills limited. They were allowed access to water ad libitum and maintained under standard conditions. The animal room was well ventilated with a temperature range of 25 - 27oC under day/night 12-12 hour photoperiodicity. The rats were randomly grouped into five groups of 5 rats each A (0mg/kg), B (150mg/kg), C (200mg/kg), D (250mg/Kg) and E (300mg/Kg).

Plant Materials

The plant material, Aspilia Africana leaves, were obtained from Okada village in Ovia North-East L.G.A of Edo State and authenticated by the botany Department, University of Benin. The leaves were sun-dried and grounded to fine powder. Crude methanolic extraction was done using 90% Methanol. The solution was filtered after 72 hours while the filtrate was concentrated to a semi solid form using the rotary evaporator, weighed and the solutions were prepared as100mg/ml and 200mg/ml respectively.

Experimental Design

The administration of the extract was totally by gavage. Proper concentrations were administered by the use of metal oropharyngeal canula and calibrated hypodermic syringe. The administration of extract was done once in a day, everyday of the week for 30 days. The control group A received no extract but 1ml of distilled water, while Groups B, C, D and E received 150mg/kg B.W, 200mg/Kg B.W, 250mg/Kg B.W and 300mg/kg B.W. of the extracts respectively. The animals were sacrificed a day after the administration of extracts stopped using the humane method.

Tissue Collection

The ovaries were dissected out by laparatomy immediately after sacrificing the animals. They were perfused in normal saline, blotted dry and weighed in electronic weighing balance. The ovaries were then fixed in 10% buffered formalin for histological assessment.

Routine Histological Preparation

The histology of the ovaries was done by a method described by Oluyemi et al (2007). The organs were fixed in 10% buffered formalin for a day after which it was transferred to ascending grades alcohol for dehydration. The tissues were passed through 50%, 70%, 90% and two changes of absolute alcohol and xylene for different durations before they were transferred into two changes of molten paraffin wax for 1 hour each in an oven at 65 oC for infiltration. They were subsequently embedded and serial sections cut using rotary microtome at 6 microns. The tissues were fixed into albumenized slides and allowed to dry on hot plate for 2 minutes. The slides were dewaxed with xylene and passed through absolute alcohol (2 Changes); 70 % alcohol, 50% alcohol and then to water for 5 minutes. The slides were then stained with Haematoxylin and Eosin. The slides were mounted in DPX. Photomicrographs were taken with x10 objective lens.

Statistical Analysis

Data were statistical compared at p values <0.001 & <0.05

Results

Figure 1

Table 1.0: Weight Of The Ovaries After The Experiment

Figure 2

Table 1.1: Weight Changes In Rats Before, During And After The Experiment

Figure 3

Figure 1.0: (Group A; 00mg/kg B.W) Photomicrograph of the ovary of control group. It shows intact ovarian tissues and few developing follicles in the cortex. The primary oocytes show no sign of alteration or degeneration. No visible active corpus luteums. Few corpora albicantia are present.

Figure 4

Figure 1.1: (Group B; 150mg/kg B.W) Photomicrograph of the ovary of group B. It shows large atretic follicles. It possesses few Graafian follicles with deranged granulosa cells. The ovarian stroma was compact with poor vascularity.

Figure 5

Figure 1.2: (Group C; 200mg/kg B.W) Photomicrograph of the ovary of group C. It shows fewer but very large atretic follicles, few Graafian follicles with deranged granulosa cells. The ovarian stroma was compact with poor vascularity.

Figure 6

Figure 1.3: (Group D; 250mg/kg B.W) Photomicrograph of the ovary of Group D. It shows large atretic follicles with compact blood vessels.

Figure 7

Figure 1.4: (Group E; 300mg/kg B.W) Photomicrograph of the ovary of Group E. It shows the presence of a few numbers of follicles with degenerated oocytes.

In general, the surface epithelia of the ovaries are not affected.

Discussion

Table 1.0 gives the wet weights of the ovaries after the experiment. It reveals a dose-dependent decrease in the experimental rats when compared with the control group. This could probably be due to the saponins and glycoside contents of this plant. This is in line with the work of Wook-Joon et al, (2003) and Subhendu et al, (2006). They have been able to point out that plant rich in saponins and glycosides cause reduction in the wet weights of uteri and dry weights of ovaries of rats.

Table 1.2 shows the weight changes in the rats before, during and after the experimental period. When the values were compared with the onset weights of the animals, there were no significant differences. This means that methanolic extract of Aspilia africana slightly reduces the deposition of fat in the animal tissues especially the adipose tissues. Continuous deposition of fat in tissues is responsible for increase in weight. This is an indicator that Aspilia africana could be used for anti-obesity purpose.

From the histology of the ovaries carried out, it is evident that Aspilia africana possesses anti-ovulatory properties as seeing in the reduction of tertiary follicles with deranged granulosa cells. These are probably due to the abundance of saponins in Aspilia africana. In a similar work done by Wook-Joon et al (2003), Korean Ginseng saponins showed similar side effects on the ovarian functions and histology of immature rats. Abnormal oocytes characterized by different types of degeneration, such as fragmentation, parthenogenesis, irregular shape or amorphous opaque mass of vitteline material and empty zona pellucida were reported after the administration of saponins. These are the effects we also observed in this work. Our results therefore justify the usage of Aspilia africana leaves, by the Mbaise and other Igbo Speaking people of Nigeria, which they boil and filter to prevent conception (Okwuonu, Personal communication). In another work, the presence of saponins, tannin, steroids, alkaloids, glycosides and terpenes has been found to possess anti-ovulatory and anti-fertility properties (Subhendu et al, 2006). Most of these compounds are present in Aspilia africana, hence its corresponding side-effects on the ovaries of experimental rats.