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The present study evaluated the abortifacient potential of aqueous-acetone extract of Adenopus breviflorus Benth Seed on the reproductive health of matured female albino rats by monitoring the indices of dysfunctions in reproductive system. Prior to the conduction of the fertility studies, the oral acute toxicity of the seed extract was evaluated for autonomic, behavioral and neurological changes, within 24 h to determine the LD50.The influence of aqueous-acetone extract of Adenopus breviflorus Benth Seed was evaluated for antioxidant, reproductive hormones and histology of uteri tissues primarily to monitor effects in female fertility. Female rats exhibiting thick clump of spermatozoa in their vaginal smear were randomly selected and used for the study to determine the abortifacient activity of the seed extract. Parameters such as number of live and dead fetuses, anogenital distance (AGD) and crown rump length (CRL), and the variation in birth weight of liters and gestation period between control and experimental animals were determined. The phytochemical composition of the seed extract was characterized by Gas Chromatography and Mass Spectrophotometry for the identification of phytochemical of toxic or therapeutic effects. Symptoms similar to clinical toxicity such as salivation, respiratory distress, weight loss and change in appearance of hair were noticed at concentrations above 1600 mg/kg BWT, there was no maternal mortality at any period of the experiment. There were changes in the behavioural, neurological and autonomic profile in groups with doses greater than 1600 mg/kg BWT. The LD50 evaluation showed that the aqueous-acetone extract of the seed was safe at dose of 5000 mg/kg BWT. The GC–MS characterization of the aqueous-acetone seed revealed isomeric derivatives of benzene-mesitylene and pseudocumene 4.28 g/100 g of sample) and 5.85 g/100 g of sample respectively as most predominant phytochemicals in the seed extract which demonstrated maternal toxicity. The effects on the female reproductive hormones of the treated animals revealed that FSH, LH and prolactin were significantly reduced (p < 0.05) in all the treated groups by the extract. Progesterone (PH) and estrogen (EH) were also reduced significantly. The study revealed scientific evidence in support of the abortifacient activity of seed extract that was significantly corresponding to the discovered phytochemical compounds.
Adenopus breviflorus Benth leaves and pods were collected from a domestic garden in Akure, Ondo State, Nigeria during sunrise and aerated until extraction. The method used for the preparation of aqueous-acetone extract of Adenopus breviflorus Benth seed was described thus, the leaves and tendril were separated from the pod and both were discarded, and the pod was broken by improvised mechanical press. Mixture was then preserved inside an air tight polythene bag to soften the endocarp and allow easy expulsion of the seed from the pod. The seeds were washed with water to remove slippery components and oven dried at 40 °C for 72 h to a constant weight. The dried seeds were then pulverized using Beltone Luinohun Blender/Miller III (model MS-223, Taipei, Taiwan). The powdered material was stocked in a sealed plastic container from which 1000 g was mixed with 1.0 L of distilled water:acetone (80:20) and stirred for 48 h at room temperature. This was then filtered with a sieve of considerable pore seize. The filtrate was concentrated using rotary evaporator and freeze drying machines to give dried residue (brownish black caked). The presence of various plant constituents in the seed extract was determined by preliminary phytochemical screening [5]. The content was concentrated to 1 mL for gas chromatography analysis and 1 μl injected into the injection port of GC. The GC equipment used was HP 6890 powered with HP chemstation Rev. A09.01 (1206) software. The split ratio will be 20:1, the carrier gas was nitrogen at inlet temperature of 250 °C with a column type of HP INNOWax and column dimensions of 30 m x0.25 mm x0.25 μm. The oven program parameters include initial temperature at 60 °C, first ramping at 12 °C/min for 20 min, maintain for 2 min and second ramping at 15 °C/min for 3 min, maintained for 8 min. The detector used FID at 320 °C at hydrogen pressure 22 psi and compressed air of 35 psi. Adult female Wistar albino rats, weighing 210−230 g were received from experimental Animal Care Center (University of Ilorin, Kwara State. Nigeria). All animals were maintained under controlled conditions of temperature (22 ± 1 °C), humidity (50–55 %) and light (12 h light/12 h dark cycle). They were acclimatized to the laboratory conditions for 14 days before the start of the experiment. Animals had free access to rat chow and drinking water. All experimental procedures were conducted in accordance with the Ethical Regulation and Guide for the Care and Use of Laboratory Animals [6]. Healthy female albino rats were deprived of food and water for 4 h and subjected to lethal dose at 50 % (LD50) and acute toxicity studies as described by Lorke, (1983) [7]. They were divided into 6 groups of 5 animals each and kept in separate cages during the experiment. The control group received food and water ad libitum. Groups 2–6 received suspension of extract of Adenopus breviflorus Benth seed orally at the doses of 10, 100, 1000, 1600, 2900 and 5000 mg/kg BWT daily. The rats were observed initially for 2 h for autonomic, behavioral and neurological changes, and for another 24 h to determine the LD50. The plant extract was tested in female albino rats for abortifacient activity as designed and reported by Khanna et al. [8]. The female rats in proestrous phase were caged with males of proven fertility in the ratio of 2:1, in the evening and examined the following day for the evidence of copulation. Female rats exhibiting thick clump of spermatozoa in their vaginal smear were separated and that day was designated as Day 1 of pregnancy. These rats were randomly distributed into 4 groups, Control group and 3 Experimental groups of 6 animals each. On Day 10 of pregnancy, animals were laprotomised under light ether anesthesia using sterile conditions. The two horns of uteri were examined to determine the implantation sites. Thereafter the abdominal wound was sutured in layers. The extract to be tested was then fed to confirmed pregnant rats, at doses of 100 (Group 2), 1000 (Group 3) and 1600 (Group 4) mg/kg body weight once daily by an intragastric (i. g.) soft rubber catheter from Day 11 up to the 15th day of pregnancy. The animals were allowed to go full term. After delivery the pups were counted and the abortifacient activity of extract was evaluated. The following parameters were computed: Number of live and dead fetuses; The anogenital distance (AGD) and crown rump length (CRL) of litters were measured by using a measuring tape. The variations in birth weight of litters and gestation period between control and experimental animal were also determined to check the abortive effect of Adenopus breviflorus Benth seed [9]. Animals in each group were completely anaesthetized and then sacrificed by cervical decapitation. The ovary and uterus were carefully removed and weighed using digital electronic balance. Blood samples were collected via cardiac puncture into non-anticoagulant tubes. The uteri and ovaries were rinsed in ice-cold 1.15 % potassium chloride solution and homogenized in 0.1 M potassium phosphate buffer (pH 7.4) by using a Teflon homogenizer. The homogenized tissues were centrifuged at 3000g for 10 min at 4 °C [6]. The sera of the control and experimental groups of female were analyzed for estrogen, progesterone, luteinizing and follicle stimulating hormone level with AccuLite master CLIA VAST Enabled kit. A Thiobarbituric Acid Reactive Substances (TBARS) assay kit (Randox) was used to measure the lipid peroxidation product MDA equivalent. One hundred microliters of homogenate was mixed with 2.5 mL reaction buffer (provided by the kit) and heated at 95 °C for 60 min. After the mixture had cooled, the absorbance of the supernatant was measured at 532 nm using a spectrophotometer. The lipid peroxidation product MDA levels are expressed in terms of nmoles MDA/mg protein using molar extinction coefficient of MDA- thiobarbituric chromophore (1.56 × 105/M/cm). The concentration of GSH was measured using standard laboratory method [10]. Homogenate was mixed with 0.2 M Tris buffer, pH 8.2 and 0.1 mL of 0.01 M Ellman's reagent, (5, 5′-dithiobis-(2-nitro-benzoic acid)) (DTNB). Each sample tube was centrifuged at 3000g at room temperature for 15 min. The absorbance of the clear supernatants was measured using spectrophotometer at 412 nm in one centimeter quarts cells. The activity of GST was measured using standard laboratory method [11]. The reaction mixture consisted of 1.0 mM GSH, 1.0 mM CDNB, 0.1 M phosphate buffer (pH 7.4) and 0.1 mL of PMS in a total volume of 3.0 mL. The change in absorbance was recorded at 340 nm by using Shimadzu spectrophotometer UV-1601 and enzyme activity was calculated as nmol of CDNB conjugate formed min−1 mg−1 protein using molar extinction coefficient of 9.6 × 103/M/cm. The activity of SOD in cells was estimated using standard laboratory method [12], with the aid of nitroblue tetrazolium as the indicator. Superoxide anions are generated by the oxidation of hydroxylamine hydrochloride. The reduction of nitroblue tetrazolium to blue formazon mediated by superoxide anions was measured 560 nm under aerobic conditions. Addition of superoxide dismutase inhibits the reduction of nitroblue tetrazolium and the extent of inhibition is taken as a measure of enzyme activity. The SOD activity was expressed as units/mg protein as compared to a standard curve. This was carried out using the manufacturer protocol of Randox Total Protein Kit [13]. 1 mL of reagent R1(Sodium hydroxide (100 mmol/l), sodium-potassium tartrate (16 mmol/l), Potassium iodide (15 mmol/l) and copper II sulphate (6 mmol/l)) was added to 0.02 mL of the test sample, the mixture was incubated at 25 °C and the absorbance was then measured against the reagent blank at a wavelength of 546 nm. All values are expressed as mean ± standard deviation. Statistical evaluation was done using One Way Analysis of Variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The significance level was set at p<0.05.
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