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.

Standard Drug Solution % inhibition of hemolysis = 100 x (OD1 – 4.5ml of standard solution consists of 2ml of OD2) / OD1

3 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells

Where OD1 is the optical density of hypotonic using standard methods [15]. buffer-saline solution alone (control) and OD2 is the optical density of test sample in Determination of serum biochemical parameters: the serum collected was analysed hypotonic solution. for total protein using biuret reaction while albumin was measured by colorimetric Procedure for evaluation of blood and estimation using the sigma diagnostics albumin serum parameters reagent (Sigma Diagnostic, U.K.) which Twenty four Wistar albino rats divided into 4 contained BromoCresol Green (BCG). groups I, II, III and IV containing 6 rats per Globulin was measured from the difference group were used for this study. The first 3 between total protein and albumin. Alanine groups were administered orally with 200, 400 aminotransferase (ALT) was measured by and 800 mg/kg body weight of the plant extract, monitoring the concentration of pyruvate while the fourth group served as control and hydrazine formed with 2,4-dinitrophenyl was administered with 3 ml/kg of distilled hydrazine while aspartate aminotransferase water. The plant extract and distilled water (AST) was determined by monitoring the were administered to the respective group concentration of oxaloacetate hydrazine orally for 28 days and blood samples were formed with 2,4-dinitrophenyl hydrazine. thereafter obtained from the anaesthetized rats through cardiac puncture for analyses. Blood Statistical Analysis samples were collected under light Ether The entire data collected was statistically anaesthesia by cardiac puncture into analyzed using one-way ANOVA and Duncan heparinized bottles for haematological studies. New Multiple Range post hoc test from SPSS Another set of blood samples were collected 16.0 package to compare the mean values of the into a clean bottle (non-heparinized) and test groups with the control. allowed to clot. The clotted blood with serum was centrifuged at 3000 rpm for 5 min. Serum RESULTS samples were collected into sterilized The results on osmotic fragility of the red blood eppendorf tubes for biochemical analysis. cells shows the extract to inhibit the hypotonic D e t e r m i n a t i o n o f h a e m a t o l o g i c a l solution induced hemolysis of the red blood parameters: Blood samples collected from cells, the extract at all the concentrations used rats into heparinized bottles were analyzed to showed an inhibition which is comparable to determine the packed cell volume (PCV), red that of indomethacin which is the standard drug blood cell (RBC) count, haemoglobin used in the study (Table I). concentration, platelet count, total and differential white blood cell (WBC) count

4 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells

Table I: Effect of aqueous leaf extract of Nelsonia canescens on hypotonic solution induced hemolysis of rat erythrocytes. The values are expressed as mean ± SEM.

Sample Concentration Optical density (OD) % Inhibition of hemolysis (%)

Hypotonic soln 50mM 0.451 ± 0.007 - Extract 2mg/ml 0.308 ± 0.021a 31.64b

Extract 4mg/ml 0.270 ± 0.101a 40.10b

Extract 8mg/ml 0.236 ± 0.071a 47.78b

Indomethacin 0.1mg/ml 0.201 ± 0.028 55.47

Superscripted items indicate significant values compared to indomethacin; a standard drug.

Table II shows the results of blood analysis as it affects erythrocytes parameters. The red blood cell count, packed cell volume and even the hemoglobin concentration does not show any significant difference from the control group.

Table II: Haematological parameters (Mean±SEM) of rats exposed to aqueous leaf extract of Nelsonia canescens Treatment RBCx106/µl PCV(%) HB(g/dl)MCV(fl) MCH(pg/cell) MCHC(%)

200mg/kg 4.8±1.4 36.5±3.1 8.8±1.3 76.04±7.3 18.33±1.8 24.11±2.1

400mg/kg 5.7±2.3 34.7±2.9 8.6±0.9 60.88±3.7 15.09±2.5 24.78±1.9

800mg/kg 5.8±2.5 37.3±1.7 8.9±0.6 64.31±4.8 15.34±3.1 23.86±1.6

Control 55.7±1.1 37.9±2.5 8.4±0.8 66.49±5.7 14.74±2.6 22.16±1.8

Control is distilled water at 3ml/kg body weight. Superscripted items indicate significant difference at p<0.05 between the test compared to control group.

The further blood analysis on total white blood cells, neutrophil and the lymphocytes shows a significant difference (p<0.05) in all the three parameters as shown in table III. The groups administered with 400mg/kg and 800mg/kg showed significant increase in total white blood cell and the lymphocytes but a significant decrease in the neutrophil count.

5 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells

Table III : Total white blood cell and differentials (Mean±SEM) of rats exposed to aqueous leaf extract of Nelsonia canescens

Treatment TWBCx103 /µl Lymp(%) Neut(%) 200mg/kg 4.8 ± 0.9 54.7 ± 6.7 43.8 ± 5.2 400mg/kg 5.7 ± 1.7a 68.1 ± 8.9b 31.9 ± 2.9c 800mg/kg 5.9 ± 0.6a 67.8 ± 3.2b 32.2 ± 4.7c Control 4.1 ± 1.2 53.9 ± 4.8 46.1 ± 1.3 Control is distilled water at 3ml/kg body weight. Superscripted items indicate significant difference at p<0.05 between the test compared to control groups.

The result of serum analysis showed a significant (p<0.05) decrease in the level of ALT for the group of rats administered with 400 and 800 mg/kg of the extract while the increase in AST is not significant. The increase in total proteins is not significant (p>0.05) for the groups administered with 200, 400 and 800 mg/kg of the extract, while the albumin:globulin ratio slightly decreased but the decrease was not significant, as shown in table IV.

Table IV: Some serum c hemistry parameters (Mean±SEM) of rats exposed to aqueous leaf extract of Nelsonia canescens

Treatment ALT(U/L) AST(U/L) T. Prot(g/l) Albu(g/l) Glob(g/l) 200mg/kg 28.1±1.4 41.3±1.7 43.2±5.7 26.1±1.3 17.1±5.3 400mg/kg 29.1±2.4a 42.9±2.0 39.9±2.1 27.7±2.7 19.5±4.1 800mg/kg 21.7±2.1a 43.0±1.7 38.2±1.1 24.3±3.1 18.7±3.2 Control 37.4±0.9 42.6±0.7 37.8±3.2 23.6±1.9 15.2±3.1 Control is distilled water at 3ml/kg body weight. Superscripted items indicate significant difference at p<0.05 between the test compared to control group.

DISCUSSION anti-inflammatory drug. It is already a known Nelsonia canescens is a medicinal plant that fact that the vitality of any cell depends on the has been acclaimed to treat many ailments integrity of its membrane [16] and exposure of including pains and inflammatory action such cell to substances such as hypotonic related diseases. In this study it was discovered solution results in lysis due to the inflow of that the extract of the plant inhibits the fluid from the region of low concentration to hypotonic solution induced rats erythrocytes high concentration inside the cell causing hemolysis in concentration dependent manner turbidity of the cell and thus the rupturing of and the hemolysis is comparable with that of the membrane of such a cell. The cell releases indomethacin which is a known non-steroidal its contents into the surrounding environment

6 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells and these results into different kinds of organs that work together to defend the body reactions such as inflammatory reactions. The against foreign substances. Lymphocytes are hemolytic effect of hypotonic solution apart one of the main types of immune cells and are from rupturing of membrane will further divided mainly into B-lymphocytes and T- render the cell more susceptible to secondary lymphocytes, while the B-lymphocytes damage through free radical-induced lipid produces antibodies through plasma cells, the peroxidation [16]. The red blood cell T-lymphocytes are programmed to recognise, membrane resembles the lysosomal respond to and to remember the antigens. It has membrane and as such, the effect of drugs on been observed that some plants contain the stabilization of red blood cell membrane different compounds and have long been used could be extrapolated to the stabilization of to modulate the humoral and cell-mediated lysosomal membrane [17]therefore, as the immune responses in Wistar albino rats [19] membrane stabilizes, it interfere with the and the result of this study corroborates the use release and or action of mediators like of Nelsonia canescens by the traditionalists as histamine, serotonin, prostaglandins, immunity booster in the treatment of some luekotrienes etc [14] which are very important diseases. The significant increase in for early phase of inflammatory reaction. neutrophils could be suggestive of the fight Substances with membrane-stabilizing against active bacterial infections but in this activities such as this extract are known for case a significant decrease was observed. their ability to interfere with the early phase of inflammatory reactions, such as the prevention The blood serum analysis showed a significant of the release of phospholipases that trigger the decrease in the activities of alanine formation of inflammatory mediators [18] aminotransferase; this enzyme is present in the thereby preventing inflammatory reactions. It liver and other cells and it is particularly useful could therefore be said that the anti- in measuring hepatic necrosis especially in inflammatory activities of Nelsonia canescens small animals [20]. Serum levels of both was due partly to the inhibition of the early alanine aminotransferase and aspartate phase of inflammatory reactions as a result of aminotransferase become elevated whenever increase in lysosomal membrane integrity. disease processes affect liver [21] and thus alanine aminotransferase is used to detect liver This study showed a significant increase in diseases [22]. Conversely, the activity of total white blood cells and the lymphocytes alanine aminotransferase in this study was and these increase observed could be observed to be significantly decreased while suggestive of increased immunity due to the that of aspartate aminotransferase remained extract. The immune system is a complex statistically unchanged. These corroborate the network of cells such as lymphocytes and use of this plant by the traditionalists as an

7 Effects of leaf extract of Nelsonia canescens on osmotic fragility of red blood cells hepatoprotective agent. The total protein, and free radicals induced DNA strand break albumin and globulin were slightly increased formation [25].Quercetol which is the flavonol but the increase was not significant. The slight extracted from the plant can prevent oxidant but not significant increase in the ratio of injury and cell death by protecting against lipid albumin to globulin suggests a slight increase peroxidation, so also, the antioxidant in the globulin production with an attendant capacities of its flavones; the apigenin and increase in IgM, IgA, and IgG which may in luteolin in free radical scavenging and against turn suggest a boost in immunity as there is no 2,2-azino-bis-(3-ethylbenzthiazoline-6- sign of hyperproteinaemia which may be due sulfunic acid (ABTS) radicals respectively to other systemic problems. These results have been reported [26]. The Caffeic acid corroborate the use of Nelsonia canescens as which is a Cinnamic acid derivative with medicinal plant by the traditionalists and Chlorogenic acid is known for its in vitro further studies on the plant could bring out antioxidant, anti-inflammatory and good remedy for different ailments as it affects antitumorigenic activities while chlorogenic humans and animals. It has been observed that acid with its protective effects on oxidative some of the phytochemical compounds stress in vivo has also been reported [27]. extracted from the plant have promising It can be concluded that the aqueous leaf medicinal potentials. Studies have shown that extract of Nelsonia canescens possesses red p-coumaric acid, a Cinnamic acid derivative blood cell membrane stabilizing effects and decreases low density lipoprotein peroxidation this may be part of the mechanism of its anti- and possesses a potential protection on cardiac inflammatory activities especially by oxidative damage induced by doxorubicin, an inhibiting the early phase of inflammatory anticancer antibiotic [23]. Cinnamic acid reactions. The extract could also boost the derivative; ferulic acid is another compound cellular immunity of the animals by increasing present in Nelsonia canescens and it has been the total white blood cell count viz-a-vis the reported to possesses a high antioxidant lymphocytes count of the rats. potential due to its resonance-stabilized REFERENCES phenoxy radical structure and this compound 1. Daniel. T.F. (2001). Catalog of has been approved in some countries as food A c a n t h a c e a e i n E l S a l v a d o r. additives to prevent lipid peroxidation as it is Contributions from the University of an effective scavenger of free radicals [24].It Michigan Herbarium, 23:115-137. 2. Essandoh, P.K., Armah, F.A., Odoi, has also been observed that normal cell J.O., Yawson, D. O. and Afrifa E.K.A. damage can be prevented in cancer patients by (2011). Floristic composition and pretreatment with ferulic acid which protects abundance of weeds in an oil palm plantation in Ghana. Journal of the cell from γ-radiation-induced damage by Agricultural and Biological Science, inhibiting peroxidation of membrane lipids 6(1): 20-31

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*Oladele G. M., Faramade I. A. and Okediran B.S. © Abuja Journal of Veterinary and Biomedical Sciences Vol. 1 April 2018 pp. 1-10

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