Abuja Journal of Veterinary and Biomedical Sciences Effects of aqueous extract of Nelsonia canescens leaf on the osmotic fragility of red blood cell and blood parameters of Wistar albino rats. *Oladele G.M.1, Faramade I.A.2 and Okediran B.S.3 1Department of Veterinary Pharmacology and Toxicology, University of Abuja, Abuja, Nigeria. 2Diagnostic and Extension Division, National Veterinary Research Institute, Vom, Plateau State, Nigeria. 3Department of Veterinary Physiology and Biochemistry, University of Ilorin, Ilorin, Nigeria ABSTRACT The herbaceous plant Nelsonia canescens is a medicinal plant used in Asian and African traditional medicine for various diseases of humans and animals. The plant has been used for the treatment of pains and inflammatory action related diseases, cancer, gout, cough, fever, cardiovascular diseases, chicken pox and even malaria. Decoction of it has also been used as immune booster in patients by the traditionalists. This study was aimed at evaluating the effects of the aqueous leaf extract of the plant on the osmotic fragility of rats' red blood cells, and also to determine the changes that occur in haematology and serum chemistry of the rats exposed orally to the extract for 28 days. Three groups of rats were administered orally with 200, 400, and 800mg/kg of the plant extract respectively while the fourth group which is the control was administered also orally with distilled water and their blood were then analyzed. The 2, 4 and 6mg/ml concentrations of the extract inhibit hypotonic solution induced rats erythrocytes hemolysis in concentration dependent manner and the inhibition is comparable to that of Indomethacin. The blood analysis showed a significant increase (p<0.05) in total white blood cells and the lymphocytes for the groups administered with 400 and 800 mg/kg of the extract while the neutrophils decreased significantly. It was then concluded that the aqueous extract of the plant inhibits red blood cell hemolysis and hence its anti-inflammatory activities. Also, the significant increase in the total white blood cells and the lymphocytes could be the reason why the plant is useful as immune booster by the traditionalists. Keywords: Nelsonia canescens, osmotic fragility, erythrocytes, rats, blood. *Corresponding email: [email protected] Tel: +234 (0)803 741 4185 This Paper was accepted on 7th November, 2017 and published 24th April, 2018 1 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells INTRODUCTION phenolic compounds such as flavonoids in the Nelsonia canescens (Lam. Spreng) otherwise polar extract; two flavones (apigenin and known as blue pussyleaf is an herbaceous luteolin), one flavonol (quercetol), four species of Acanthaceae family. It is a small cinnamic acid derivatives (p-coumaric acid, perennial herb with soft decumbent villous caffeic acid, chlorogenic acid and ferulic acid) branches; creeping, sprawling or prostrate and one benzoic acid (gentisic acid) derivative plant that often grows in disturbed and open were identified [13]. Phenolic compounds are habitats where it can be weedy [1]. The plant is important constituents of the human diet and also known as agricultural weed as it grows in they have been recognized largely as beneficial rice and oil palm plantations thereby reducing antioxidants, antibacterial and enzyme crop yield by competing with crop plants for inhibitors. This study was to determine the common resources [2], it is a very agile effects of aqueous extract of the leaf of the dispenser thereby having high dispersal ability plant on osmotic fragility of red blood cell and and thus distributed across the tropics[3]. blood parameters including serum chemistry. Nelsonia canescens is a native to Africa, Asia and Australia [4] where it is also reported to be MATERIALS AND METHODS used as cover crop to suppress the growth of weeds in banana plantations as it invades large Plant Materials areas of the plantation with no visible adverse The fresh samples of Nelsonia canescens were effects on the banana crop but limiting the collected from Ologun eru area of Ibadan in Ido possibility of other weeds to invade [5]. The Local Government area of Oyo State, Nigeria plant is a medicinal plant used in African and where it grows as weed. The confirmatory Asian traditional medicine; for the treatment of identification of the plant was done at Forest fever and as an analgesic in cold, flu, cough and Reserve Institute of Nigeria (FRIN) Ibadan. viral infections in Africa, use in Indian The leaves were plucked, washed with water to traditional medicine to treat pain and remove dirt and then shade-dried at room inflammation [6], the plant is also used in the treatment of cancer, gout, cardiovascular and temperature. After drying, the leaves were inflammatory diseases [7]. The traditional milled into powder using mortar and pestle and healers use the root, fruit and leaves of the plant then stored in an air tight container for for different disease conditions [8].The root is extraction. used by traditional healers of Gandhamardan hills of Orissa in the local name of Badarasna Extraction of the Leaves (Rasna) for the management of pain and The cold maceration extraction method was inflammatory conditions such as arthritis, employed in which 250g of the powdered cutting wound, and bone fractures [9]while the whole plant is used for its hepatoprotective sample of the leaves was added to 1 liter of action [10].Nelsonia canescens grows fast in distilled water and allowed to stay for three Western part of Nigeria where it is used for the days with intermittent shaking. Thereafter, the treatment of various ailments. The traditional mixture obtained was filtered using filter paper practitioners in this part of the country use the and the filtrate which is the extract was plant for the treatment of malaria [11], pain, chickenpox, constipation and gastric ulcer; the concentrated using vacuum rotary evaporator patient is required to take a full cup of the plant (IKA, Germany) at an optimum temperature of decoction at least three times daily or take after 500C. The percentage yield of the extract was meal instead of water [12], they also use 5.77%. A fresh 10% (w/v) solution of the decoction of the plant to boost the immunity of extract was prepared with normal saline to patients. The phytochemical analysis of Nelsonia canescens shows the presence of make appropriate dosage required for the studies. 2 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells Animals for the study hypotonic saline (0.25%w/v); 1ml of Adult Wistar albino rats of both sexes weighing p h o s p h a t e b u ff e r ( p H 7 . 4 ) ; 1 m l o f 120–175g, obtained from National Veterinary Indomethacin (0.1mg/ml) and 0.5ml rat red Research Institute Vom, Plateau State Nigeria, blood cells in isotonic saline. were used for the study. The animals were housed in steel metal cages at the Animal Procedure for osmotic fragility test House of the Faculty of Veterinary Medicine, The method used was as described by Shinde et University of Abuja and acclimatized for two al in 1999 [14]. Whole blood was obtained weeks before the experiments. The animals from rats through cardiac puncture into a were given free access to water and fed with heparinized tube, centrifuged and supernatant growers mash bought from the local market. was carefully pipetted. The remaining packed cells was washed four times with equal volume Solutions for osmotic fragility test of isotonic buffer solution (154 mM NaCl in 10 Standard Drug mM sodium phosphate buffer; pH 7.4), the 0.10mg/ml of Indomethacin was prepared in packed cells were been centrifuged each time isotonic saline (0.85% NaCl) to make the at 1000 rpm for 10 minutes. 10% rat concentration required for the study. erythrocytes suspension was prepared with normal saline and kept in the refrigerator at 40C Red Blood Cell Suspension as stock erythrocytes. 10% (v/v) of rat red blood cell suspension was 1 ml of varying concentrations of the extract (2, prepared with normal saline and kept in 4 or 6mg/ml) or 0.10 mg/ml of indomethacin in refrigerator at 40C as stock erythrocytes and the the case of standard drug solution, was mixed osmotic fragility activity of the extract on the with 1 ml of phosphate buffer, 0.5 ml of stock red blood cells was assessed using hypotonic erythrocytes and 2 ml of the hypotonic solution solution-induced rat erythrocyte hemolysis. was added to make 4.5 ml. The test control Test Solution consists of 2ml of hypotonic saline 4.5ml of test solution consists of 2ml of (0.25%w/v), 1ml of phosphate buffer (pH 7.4), hypotonic saline (0.25%w/v); 1ml of 1ml of isotonic saline and 0.5ml of rat red phosphate buffer (pH 7.4); 1ml of test extract blood cells in isotonic saline, the reaction (1mg/ml - 8mg/ml) in normal saline and 0.5ml mixtures were incubated at 37°C for 30 of rat red blood cells in isotonic saline. minutes, centrifuged for 10 minutes at 1000 rpm and the absorbance of the supernatant Test Control s o l u t i o n w a s m e a s u r e d w i t h 4.5ml of test control consists of 2ml of spectrophotometer at 540 nm. Each hypotonic saline (0.25% w/v); 1ml of experiment was carried out in triplicate and the phosphate buffer (pH 7.4); 1ml of isotonic average was taken. The percentage inhibition saline and 0.5ml of rat red blood cells in of hemolysis or membrane stabilization was isotonic saline calculated according to modified method described by Shinde et al in 1999.