Toxicological Study of Hydroalcohol Leaf Extract of Acanthospermum hispidum ( Asteraceae ) 1

Corresponding Author: Diallo Aboudoulatif Department of Toxicology, Faculty of Health Sciences, University of Lome-Togo, Togo Email: aboudoulatif@yahoo.fr Abstract: Acanthospermum hispidum is a widely plant used in African traditional medicine. This study aims to assess cardiotoxicity and subchronic (28-days) oral toxicity of hydroalcohol leaf extract of A. hispidum. The sub-chronic toxicity was evaluated after administering daily oral doses of 500 and 1000 mg/kg body wt., for 28 days to the rats, biochemical and haematological assessments as well as body and relative organ weights of the rats were carried out. The results of the study have shown that blood sodium was significantly (p < 0.05) lower in the group treated with 500 and 1000 mg/kg of A. hispidum extract and the blood platelet was significantly high. The weekly body and organ weight of the rats showed no significant differences between the control and the rats treated with the extract except for the testis where there was a significant decrease (p < 0.05) in rats that received 1000 mg/kg, i.e., 1.16±0.2 g against 0.78±0.22 g for the control. A discreet seminiferous tubular atrophy and fibrosis of the tube wall were observed at this dose. The hydroalcoholic extract of A. hispidum at 0.1; 1; 10 and 100 mg/ml on toad heart and isolated atria of guinea pigs have caused a significant increase in cardiac amplitude (positive inotropic) and a significant decrease (p < 0.0001) in frequency (negative chronotropism). Our results suggest that the hydroalcohol extract of A. hispidum is relatively toxic to the testis and caution must be taken at high doses especially for the heart.


Introduction
Acanthospermum hispidum is an annual plant belonging to Asteraceae family. It is widespread in the tropics and among the weeds. It is a plant used in traditional medicine in treating of a lot of diseases. The plant is used in the treatment of yellow fever, malaria and stomach disorders (Mann et al., 2003;Chakraborty et al., 2012;Agbodeka et al., 2016). In some parts of South America, it is used as diuretic. In Ivory Coast, it is use for its many therapeutic properties such us anti-malarial, anti-hypertensive, antispasmodic, anthelmintic and abortion (Yapi, 2013). In Togo, A. hispidum is used in the treatment of headache, malaria, typhoid fever and hemorrhoids.
2 Thus, the present study has the objective to study the cardiotoxicity and sub-chronic (28-days) oral toxicity of hydroalcohol leaf extract of A. hispidum.

Extraction of Acanthospermum hispidum
The study was conducted at the Faculty of Health Sciences and the Faculty of Science of the University of Lome, Togo. A. hispidum leaves were harvested at Adidogomé (Togo) in October 2017. They were identified by Professor K. Akpagana of the Department of Botany at the University of Lome (Togo) and a specimen was preserved in the herbarium of the Laboratory of Botany and plant Ecology at the Faculty of Science in the reference TOGO15380. The leaves were dried protected from light and reduced to powder. The powder (750 g) was soaked of water-ethanol 95% (50/50: v/v). The mixture was stirred mechanically for 72 hours. The macerate was filtered. The filtrate was evaporated in using a Rotavapor R 210. The yield of this extraction was 16.5%.

Experimental Animals
Male Wistar rats (199.70±6.28 g) were provided by the department of animal physiology of the Faculty of Science of the University of Lome, and used for toxicological tests. Toads (Bufo marinus) ranged in body weight from 90-160 g (mean 120 g) were captured in night, in damp places and were used for the study of cardiac activity ''in vivo''. The guinea pigs were supplied by the breeding farm of Albatross in Lome (Togo) and used for contractile activity on isolated atria. All these animals were housed in environmental normal conditions and fed standard diets and water.

Phytochemical Screening
Plant materials were screened for the presence of alkaloids, saponins, tannins, total phenols, anthraquinones, flavonoids, sterols and cardiac glycosides using the methods previously described by Tona et al. (1998).

Sub-Chronic Toxicity Study
The repeat dose of oral toxicity study was carried out according to OECD guideline 407 (2008). Three groups (T, D1, D2) of 6 male rats were formed. The control group received distilled water. The groups D1, D2 received the hydroalcohol extract of A. hispidum leaves at 500 and 1000 mg/kg body weight respectively. Extract was administered daily for 28 days at the same time. The animals were observed at least twice daily for morbidity and mortality. Body weight of the animals was evaluated daily. At the 29th day, after an overnight fast, rats were anaesthetized with ether and blood sample for haematological and biochemical analysis was collected into tubes with or without EDTA respectively. Haemoglobin (Hb), haematocrit (Ht), Red Blood Cells count (RBC), White Blood Cells count (WBC), Mean Corpuscular Haemoglobin Concentration (MCHC), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Volume (MCV) and platelet count were determined using automatic counter Sysmex (K21, Tokyo, Japan). Biochemical analysis was performed in serum obtained after centrifugation of total blood without anticoagulant, at 2500 rpm for 15 min. Standardized diagnostic kits (Labkits) and a Biotrons spectrophotometer were used for spectrophotometrical determination of the following biochemical parameters: Alanine aminotransferase (ALT), Aspartate Aminotransferase (AST), creatinine, Alkaline Phosphatise (ALP), Glucose (Glu), total proteins, γGT and urea. Necroscopy of all animals was carried and the organ weights (heart, testis, colon, liver, kidney and spleen) were recorded. Each weighed organ was then standardized for percentage body weight of each rat (relative organ weight). Histological study of organs was done after sacrificing the animals on 29th day. Organs were removed, fixed in 10% formaldehyde, embedded in paraffin and sectioned at 5 mm. Tissue sections were stained with haematoxylin eosin (H and E) for general morphology.

Cardiac Activity Study in Toad
After toads demedullation, a midline ventral incision was made and the pectoral girdle and xiphoid process were removed to expose the heart and major vessels. The apex of the heart with suspended in a fine wire bonded to the transducer coupled to a PowerLab recording system and an application software LabChart all ADI Instruments, listing the frequency and amplitude heart. The software was always calibrated before each experiment. The different doses of the extract and digoxin were administered directly to the heart in vivo. Each test was repeated 5 times.

Contractile Activity on Isolated Guinea Pig Atria
The guinea pigs were sacrificed by cervical dislocation after ether anesthesia. The atria were quickly removed and placed in Mac Ewen physiological solution (NaCl: 15 g; KCl0.24 g; CaCl: 0.48 g; PO4H2Na: 0.29 g; CO3HNa: 2 g; MgCl2: 0.1 g; Glucose 4 g) at 37°C. The 3 atria were then mounted into the vessel body. The right atrium was attached to the hook which is situated at the bottom of the tank and the left atrium is connected to the transducer. The body under a base tension of 1 g was bathed in the physiological Mac Ewen solution maintained at 37°C and aerated with pure oxygen. The atria were washed several times just after installation. After a few minutes of equilibration, cumulative concentrations of 0.1 mg/mL; 1 mg/mL; 10 mg/mL; 100 mg/mL of extract was tested on the amplitude and frequency of contractions. The organ is connected to the transducer coupled to a PowerLab recording system and application software LabChart all ADI Instruments, recording the frequency and amplitude of heart. The effect of each dose on the atria was recorded for 10 minutes. The same operation was repeated three times with each dose.

Statistical Analysis
The software GraphPad Prism 6.02 (Graph Pad Software, Inc. USA) was used to analyze our results. These were expressed as mean value accompanied by the standard error of the mean (m ± SEM). The number (n) of value was specified in each case. The Analysis of Variance (ANOVA) was used to compare different groups. The difference between two groups was determined using the Tukey test. The significance level was set at p < 0.05.

Phytochemical Screening
The phytochemical screening was first performed; it showed the presence of traces of alkaloids, flavonoids, saponins, tannins and cardiac glucosides.

Sub-Chronic Toxicity
The hydroalcohol extract of A hispidum leaves resulted in no significant change in body weight of rats of different groups (Table 1). But there is a significant decrease (p < 0.001) in testis relative weight to the dose of 1000 mg/kg ( Table 2).
The extract at 500 and 1000 mg/kg significantly (p < 0.001) increased platelet counts after 28 days (Table 3). The Table 4 shows the biochemical analysis. A. hispidum significantly (p < 0.0001) the decreased blood sodium level. No significant damage was observed in rat liver, kidney, spleen and heart when compared with control. The histological sections of testis have shown a seminiferous tubule atrophy and fibrosis of the tube wall (data was not shown).

The Effect of A. hispidum on the Toad Heart
The hydroalcohol extract of A. hispidum at 0.1; 1; 10 and 100 mg/mL caused an increase in toad cardiac amplitude (positive inotropic) and a significantly decrease (p < 0.0001) the heart rate (negative chronotropism) ( Table 5).

The Effect of A. hispidum on Guinea Pig Isolated Atria
The extract at concentrations of 0.1 mg/ml and 1 mg/ml on isolated guinea pig atria, resulted in an increase of the amplitude (positive inotropic) and a significant decrease (p < 0.0001) of the frequency (negative chronotropic) ( Table 6). At concentrations of 10 mg/mL and 100 mg/mL the extract caused a cardiac arrest.

Discussion
The phytochemical screening showed the presence of alkaloids, flavonoids, saponins, tannins and cardiac glucosides. These chemicals would be involved in the different biological activities of this extract. Our result confirms those of Roy et al. (2010), which have already noted the presence of these chemical groups in the leaves of A. hispidum. Edewor and Olajire (2011), have noted the lack of alkaloids and cardiac glycosides in the aerial parts of A. hispidum. Our result has noted the lack of anthracene in our extract. This result confirms those of Edewor and Olajire (2011), which also noted the lack of anthracene.
The hydroalcohol extract of A hispidum leaves induced no significant change in body weight of rats of different groups. The administration of hydroalcohol extract of A hispidum leaves to rats resulted in no significant change in body weight of rats of different groups, but there is a significant decrease (p < 0.001) in testis relative weight at the dose of 1000 mg/kg. The assessment of the weight of organs such as the liver, kidney, spleen, testes, heart, pancreas, brain and language is very important in toxicological studies. The weight of a body or more, the relative weight is an important index used in physiology and toxicology (Dybing et al., 2002). The decrease in testis relative weight translates a testicular atrophy. The same result 5 was reported by Ali and Adam (1978). This reduction in the relative weight of the testis was confirmed by the histological sections of testis showing a seminiferous tubule atrophy and fibrosis of the tube wall.
Achieving Blood Counts (CBC) is also very important in toxicological studies. The hematopoietic system is one of main targets of toxic substances and consequently, an important parameter of the physiology of the human or animal (Olson et al., 2000;Diallo et al., 2008). The extract at 500 and 1000 mg/kg significantly (p < 0.0001) increased platelet counts after 28 days. The thrombocytosis can be attributed to increased production and secretion of thrombopoietin, the major regulator of platelet production (Kaushansky, 1995).
The measurement of some biochemical parameters such as enzyme activities in tissues and body fluids plays a major role in the study of disease, diagnosis and assessment of toxicity. The liver, kidneys and lungs are the main organ affected by the metabolic reaction caused by toxic substances. The liver is the organ most affected because it is the most important organ of detoxification. Our result has shown that A. hispidum significantly (p <0.0001) decreased blood sodium. This hyponatremia may be due to the diuretic effect of this extract hence its use as an antihypertensive .
On toad heart, A. hispidum has increased the cardiac amplitude (positive inotropic) and a significantly decrease (p < 0.001) the cardiac frequency (negative chronotropism). These effects may be due to cardiac glycosides contained in A. hispidum leaves.
The contractile activity on isolated guinea pig atria with A. hispidum leaves extract resulted in an increase of the amplitude (positive inotropic) and a significant decrease (p < 0.0001) of the frequency (negative chronotropic). These effects are similar to the effect of cardiac glycosides which are also present in our extract. Cardiac glycosides increase the contractile force (positive inotropic effect) by increasing the intracellular calcium during systole. They bind to a specific site of the Na-K-ATPase. Depolarization occurs at the opening of fast sodium channels. In addition, calcium flux induces the release of calcium from the sarcoplasmic reticulum, producing contraction (Sellers et al., 2007).

Conclusion
After this work, A. hispidum essentially contains alkaloids, flavonoids, saponins, tannins and cardiac glycosides. He induced a significant decrease in relative testes weight, blood sodium and increased platelets. Cardiac disorders were observed, characterized by an increase in the force of contraction, a significant decrease in the frequency and increased heart muscle tone, due to the presence of cardiac glycosides.