INTRODUCTION

Scoparia dulcis L. (Scrophulariaceae) is a widespread herb in tropical and subtropical regions where it is well known as a folk-medicinal plant¹.

Previous investigations evidenced a number of its medicinal properties against diabetes, inflammation and oxidation in vivo^2,3 and its impact on lipid peroxidation^4,5. Moreover, its protective role on insulinoma cell line RINm5F⁶ and on kidney, heart and liver of rats exposed to cadmium² were demonstrated. A few studies were carried on the hepatoprotective role of the plant^7-10. Along with its protective effect on liver and kidney, S. dulcis is used to help with the management of memory impairments in local populations in Burkina Faso¹¹ and our previous research related its positive impact on brain protection in vitro¹².

Indeed, the brain and nervous system have limited antioxidant capacity and are most vulnerable to oxidative stress¹³. Acetylcholine (Ach) is a neurotransmitter in neuronal cells and its deficiency in cerebral cortex was found to be a major cause of neurodegenerative disorder such as Alzheimer’s disease¹⁴. Its action is stopped by acetylcholinesterase (AChE), a key enzyme catalyzing the hydrolysis of acetylcholine (ACh) in the nervous system of animals and insects. The use of AChE inhibitors can help for neuroprotection in neurodegenerative disorders by enhancing the Ach level in the brain¹⁵.

Previous study stated a markedly inhibition of acetylcholinesterase by scoparia dulcis in vitro ¹².

This study was then carried out to contribute to the understanding of the in vivo protective role of Scoparia dulcis particularly on brain based biomarker AChE activity along with the assessing of its hepatoprotection and renoprotection on rats exposed to carbon tetrachloride toxicity.

MATERIAL AND METHODS

Plant material: The whole plant of Scoparia dulcis L. was harvested around the dam of Loumbila in August, 2011. The plant was authenticated by the Plant Ecology and Botany Laboratory/University of Ouagadougou where a specimen was deposited in the herbarium under the identification code SD_ac01. The sample was dried at room temperature (25°C) and then reduced to powder and kept for extraction.

Extraction: A mass of 25 g of plant powder was macerated for 24 h in 250 mL of a mixture constitute by acetone and water (80/20, v/v). After filtration, the acetone was evaporated in a rotavapor apparatus and the aqueous phase obtained was lyophilized to constitute the extract¹².

Experimental design
Acute toxicity: The toxicity of the plant extract was evaluated according to the protocol described by OECD¹⁶. A group of 3 Wistar rats was used and fasted for 18 h. Each rat then received a single dose of 3000 mg kg^–1 of body weight orally. They were then observed constantly during the 1st 30 min and then regularly for the next 24 h to note any sign of toxicity (contortions, panic, moribund state, death .). The observation continued beyond 14 days after which the animals were sacrificed and buried according to the rules of ethics.

Animals treatment conditions: The animals were treated based on previous study by Murthy et al.¹⁷ with slight modifications.

Wistar female rats (223-302 g), 3 months old, were acclimated at 25°C with free access to water and food. They were then divided into 5 groups of 6 rats each. Each group receives for 7 days, a single daily dose of carboxyl methylcellulose (CMC, 0.5%) as vehicle alone, either silymarin (100 mg kg^–1 b.wt.) or the extract of Scoparia dulcis at the respective doses of 200 and 400 mg kg^–1 as follow:

After 7 days of treatment, the rats of all groups (except those in group 2) are intoxicated by intra-peritoneal injection with carbon tetrachloride (2 mL kg^–1 b.wt.). About 24 h later, the rats are asleep under the anesthetic effect of diethyl ether administered by inhalation. The blood is then collected by cardiac puncture using a sterile syringe and stored in 2 types of sterile tubes containing EDTA (acide Ethylène Diamine Tetra Acetique) or not. The liver, kidneys and brain are also collected for the preparation of homogenates (5%) by grinding the organs in a solution of potassium chloride (KCl, 0.15 M) followed by centrifugation at 4000 rpm for 10 min. The collected supernatant constitutes the homogenate.

Quantification of biochemical parameters: The serum obtained by centrifugation of the tubes without EDTA at 1500 rpm is used to determine the biochemical parameters.

Alanine aminotransferase (ALT or GPT), aspartate aminotransferase (AST or GOT), triglycerides, HDL-cholesterol, uric acid and urea are quantified using a kit (LABKIT) according to the manufacturer’s instructions.

Creatinine is quantified using the spinreact kit according to the manufacturer’s instructions.

Total proteins are assayed from liver, kidney and brain homogenates using the spinreact kit according to the manufacturer’s instructions.

Superoxide dismutase estimation: Superoxide dismutase (SOD) was assayed as described by Murthy et al.¹⁷. It is based on the reduction of nitroblue tetrazolium (NBT) in blue formazan crystals. Briefly, the liver or kidney homogenate (0.5 mL) was mixed with 1 mL of sodium carbonate (125 mM), 0.4 mL of NBT (24 μM) and 0.2 mL of EDTA (0.1 mM). The reaction is then initiated by the addition of 0.4 mL of hydroxylamine hydrochloride (1 mM). After 5 min of incubation at 25°C, the absorbance is measured at 560 nm against a blank without hydroxylamine hydrochloride. A control without homogenate is prepared under the same conditions. One unit of SOD activity is the amount of enzyme that inhibits 50% of NBT. The specific activity of SOD was expressed as units per milligram of protein.

Lipid peroxidation: Peroxidation of lipids was evaluated as described by Murthy et al.¹⁷. It is based on the reaction between thiobarbituric acid (TBA) and malondialdehyde (MDA) to form a detectable chromogen at 532 nm. A volume of 0.5 mL of homogenate of liver or kidney is mixed with 1 mL of a solution of potassium chloride (KCl, 0.15 M). Peroxidation is then initiated by adding 250 μL of iron tri-chloride (FeCl₃, 0.2 mM) and then incubated at 37°C for 30 min. The reaction is then stopped by adding 2 mL of an ice-cold mixture of a hydrochloric acid solution (HCl, 0.25 N) containing 15% trichloroacetic acid, 0.3% thiobarbituric acid and 0.05% ascorbic acid. Then the mixture was incubated in a hot bath at 80°C for 60 min. After centrifugation at 4000 rpm for 10 min, the absorbance was read at 532 nm. Lipid peroxidation measures the reactive substances of thiobarbituric acid (TBARS) in MDA equivalent using a molar extinction coefficient of 1.56×10⁵ M^–1 cm^–1. Results are expressed in nmole mg^–1 of protein.

Acetylcholinesterase activity: This method was based on that described by Ellman et al.¹⁸ with some modifications. The reaction mixture was constituted by 300 μL of buffer (Tris-HCl 50 mM pH 8.008, 0.1% BSA) and 100 μL of brain homogenate. About 500 μL of DTNB (3 mM) and 100 μL of ATCI substrate are then added and the kinetics was monitored at 405 nm on a spectrophotometer for 5 min against a blank without homogenate. The activity of acetylcholinesterase (AChE) is expressed by Beer Lambert’s law using a molar extinction coefficient of 13.6 mM^–1 cm^–1 in μmole min^–1 mg^–1 of protein.

Statistical analysis: All the reactions were performed in triplicate and data are presented as mean±standard deviation. Data were analyzed by one-way analysis of variance followed by the Tukey multiple-comparison test. Analysis were done by using XLSTAT7.1 software. A p-value <0.05 was used as the criterion for statistical significance.