2 Pharmacology Dept., Bayero University, Kano. Acute and Sub-chronic Toxicity Studies of the Methanol Leaf Extract of Dalbergia saxatilis (Hook.F) in Albino Rats 1128 Fatima Ismail Hassan1, Abdulkadir Umar Zezi1, Abdullahi Hamza Yaro2, and Umar Habib Danmalam3 1 Dept. of Pharmacology & Therapeutics, Ahmadu Bello University, Zaria. 2 Pharmacology Dept., Bayero University, Kano. 3 Dept. of Pharmacognosy & Drug Development, Ahmadu Bello University, Zaria. INTRODUCTION RESULTS DISCUSSION Table 5 :Effect of methanol leaf extract of D. saxatilis on liver function indices Acute Toxicity Studies The median oral lethal dose for the rats was found to be greater than 5000mg/kg. Sub-chronic Toxicity studies Table 1:Effect of methanol leaf extract of Dalbergia saxatilis on Average body weight (g) Table 2:Effect of methanol leaf extract of D. saxatilis on Organ weight ratio The decoction of the leaves of Dalbergia saxatilis is used in Traditional Medicine for various ailments such as cough, small pox, skin lesions, bronchial ailments and toothache (Saha et al., 2013). However, its toxicity has not been established scientifically. The objective of the study was to conduct acute and sub-chronic toxicity studies of the methanol leaf extract of Dalbergia saxatilis (Hook. F) in albino rats. There were statistical differences observed in the 250mg/kg, 500 mg/kg and 1000 mg/kg groups of the extract compared to the control for average body weight at P< 0.05 and P< 0.01. However, there were no differences observed in all the organs in both control and treatment groups. A statistically significant decrease was observed in WBC count at P< 0.01, P< 0.001 and P< 0.01 in all groups of the extract respectively, this may be due to decreased production of total white blood cells in the bone marrow or increased destruction of the cells, and it indicates bone marrow depression which occurs as a result of viral infection or toxic reactions (Goerge-Gay and Parker, 2003). A significant reduction in sodium, chloride and bicarbonate concentrations at P< 0.05 was observed. Levels of sodium, chloride and bicarbonate is a good indicator on how well the kidneys and heart are functioning. Electrolyte imbalance may be result from kidney failure (Briggs et al, 1996; Halperin and Goldstein, 1994). A statistically significant increase in ALT was observed in the 250mg/kg and 500mg/kg groups of the extract at P< 0.05 and for AST in the 250mg/kg group of the extract at P< 0.05 respectively. Elevation in the levels of AST and ALT may be as a result of hepatocellular injury (Giboney, 2005). An elevated level of ALP may be as a result of injury to liver, bone, leukocytes, kidneys, intestine or placenta (Hall and Cash, 2012). Hispathological leisons were observed in the organs (Table 6) especially in the liver and kidney (Plate 1-8). Group Alanine transferase (U/L) Aspartate transferase Alkaline phosphatase Serum bilirubin (mg/dL) Control 62.36 ± 11.657 181.22 ± 34.639 225.40± 10.605 1.29 ± 0.191 250mg/kg 119.48 ± 20.239a 350.23 ± 29.491a 362.25 ± 50.579 0.94± 0.147 500mg/kg 107.00 ± 6.909a 261.38 ± 24.226 311.60 ± 15.667 0.99 ± 0.182 1000mg/kg 95.34 ± 6.585 278.80 ± 51.923 341.25 ± 66.386 0.72 ± 0.107 MATERIALS AND METHODS Treatment groups Dosemg/kg Week 0 Week 1 Week 2 Week 3 Week 4 Distilled water 10 ml/kg 119.0±7.0 134.1±6.9 138.6±5.2 146.5±3.2 146.1±3.7 Extract 250 115.8±5.7 117.8±6.4 116.3±5. 1a 119.6±9. 1a 128.3±4.8 500 116.2±5.1 110.1±3.1a 118.1±2.9 127.0±6.8 140.3±6.0 1000 116.6±4.1 111.3±4.3a 109.0±10. 0b 121.7±8.7 123.2±10.8 Preparation of Plant Extract The leaves collected were identified, air-dried, powdered and extracted by cold maceration using absolute methanol. The extract was evaporated to dryness under reduced pressure and controlled temperature (50°C). Study Animals Wistar rats of both sexes weighing 100-150g were used. Acute Toxicity Studies This was carried out as described by Lorke (1983). Sub-chronic Toxicity Studies The study was carried out in accordance to WHO (1992) and OECD (1995) guidelines. Twenty rats of either sex were deprived of food for 24 hours, and divided into four groups of five rats each. Group 1 received distilled water, while rats in groups 2, 3 and 4 received graded doses of the extract (250 mg/kg, 500 mg/kg and 1000 mg/kg) body weight respectively daily for 28-days. The rats were allowed free access to food and water throughout the duration of the experiment and were observed daily for general symptoms of toxicity and mortality. Rats were then sacrificed on the 29th day of the experiment by cervical dislocation. Blood samples were collected, and the heart, spleen, stomach, liver and kidney were removed. Calculation of organ weight ratio The organs removed were weighed for determination of relative organ body weight ratio. Biochemical and Haematological studies Blood samples were collected into plain and EDTA bottles for evaluation of alanine aminotransferase(ALT),aspartate aminotransferase (AST) and alkaline phosphatase (ALP), and serum bilirubin , serum urea nitrogen, creatinine, chloride, sodium, potassium, and bicarbonate using commercial kits obtained from Reckon Diagnostics P. Ltd, India. Packed cell volume (PCV), haemoglobin concentration (Hb), platelets (PLT), white blood cell count (WBC) and differentials, mean corpuscular haemoglobin concentration (MCHC), using an automated haematological machine (Cell-DynTM Abbot, US). Histopathological studies The organs removed were fixed in 10% formal saline for at least 48 hours, and processed routinely, the tissues were embedded in paraffin wax. Histological sections were cut at 5 - 6 μm and stained with routine haematoxylin and eosin. Photomicrographs of representative lesions were taken at various magnifications. Statistical analysis Data were analysed using one way analysis of variance followed by Poc Dunnett t-test and Bonferonni test. ‘a and b’ in superscript represent P< 0.05 and P< 0.01, level of significance respectively. Values are ± SEM, n= 5. Table 6 :Effect of methanol leaf extract of D. saxatilis on selected organs Tissue Control 250mg/kg 500mg/kg 1000mg/kg Liver Unremarkable Vascular congestion with slight lymphocyte hyperplasia Slight lymphocyte hyperplasia Slight peri-vascular necrosis Kidney Massive glomerular necrpsis with tubular distortion Glomerular necrosis with slight tubular distortion Intense tubular and glomerular necrosis Spleen Moderate lymphocyte hyperplasia Intense lymphocyte hyperplasia Stomach Damaged mucosa Heart ‘a and b’ in superscript represent P< 0.05 and P< 0.01, level of significance respectively. Values are ± SEM, n= 5. Organs Control 250mg/kg 500mg/kg 1000mg/kg Heart 4.208 x 10-3± 0.0004 2.037 x 10-1± 0.199 4.044 x 10-3±0.0003 2.038 x 10-1±0.199 Stomach 1.041 x10-2± 0.0006 2.092 x 10-1± 0.198 1.107x10-2± 0.0009 2.094 x 10-1± 0.198 Spleen 7.458 x10-3± 0.0009 2.050 x10-1± 0.199 5.169 x10-3± 0.0004 2.045 x10-1± 0.198 Kidney 6.939 x10-3± 0.0005 2.057 x10-1± 0.198 7.287 x10-3± 0.0001 2.057 x10-1± 0.199 Liver 3.361 x10-2± 0.0028 2.279 x10-1± 0.193 3.408 x10-2± 0.0011 2.269 x10-1± 0.193 CONCLUSION These findings provide evidence for the relative acute safety of the extract, but toxic on prolonged use, as manifested in liver, kidney, spleen, and stomach damage. Plate 2: Rat liver showing vascular congestion with slight lymphocyte hyperplasia. Mag(X 250) after 28days treatment with 250mg/kg of the methanol leaf extract of D. saxatilis Values are ± SEM, n= 5. there were no statistical differences observed in all organs. Plate 1: Rat liver showing normal hepatocytes. Mag (X 250) after 28 days treatment with distilled water 10 ml/kg REFERENCES Table 3: Effect of the methanol leaf extract of D. saxatilis on Haematological indices Lorke D. (1983). A New Approach to Practical Acute Toxicity Testing. Archives of Toxicology; 54: 275-287. OECD. (1995). Repeated Dose 28-day Oral Toxicity Study in Rodents. OECD guideline for testing of chemicals 407: 1-8. Saha S., Shilpi J.A., Mondal H., Hossain F., Anisuzzman M., Hasan M., Cordell G.A. (2013). Ethnomedicinal, phytochemical, and pharmacological profile of the genus Dalbergia L. (Fabaceae). Phytopharmacology; 4: 291-346. WHO (1992). Research guidelines for evaluating the safety and efficacy of herbal medicine. WHO regional office for western pacific Manila, Philippine p. 38. Hall P, and Cash J. (2012). What is the Real Function of the Liver ‘Function’ Tests? Ulster Medical Journal; 81(1):30-36 Halperin M.L., and Goldstein M.B. (1994). Fluid, Electrolyte, and Acid-Base Physiology: A Problem-Based Approach. 2nd ed. Philadelphia, PA: W.B.Saunders. Goerge-Gay B., and Parker K. (2003). Understanding the Complete Blood Count With Differential. American Society of PeriAnaesthesia Nurses; 18(2): 96-117. Goldstein, R., and Schnellman R. (1996). “Toxic responses of the kidney,” in Casarett and Doull’s Toxicology: The Basic Science of Poisons, 5th ed., Klaassen C.D., ed., McGraw-Hill, New York, pp. 417–442. Giboney P.T. (2005). Mildly elevated liver transaminase levels in the asymptomatic patient. American Family Physician; 71:1105-10. Parameters Control 250 mg/kg 500 mg/kg 1000 mg/kg PCV (%) 33.88 ± 0.926 32.28 ± 0.576 31.04 ± 1.427 34.38 ± 2.527 Hb (g/dL) 130.80 ± 3.774 125.00 ± 2.273 121.00 ± 6.419 131.75 ± 10.379 WBC(x 109/L) 14.04 ± 1.129 8.08 ± 1.508 b 6.10 ± 1.423 c 6.68 ± 0.342 b MCHC (g/L) 386.00 ± 5.206 386.75 ± 2.496 389.00 ± 3.755 382.25 ± 3.881 PLT (x 109) 698.00±63.234 544.00±52.857 597.20±70.532 633.75 ± 83.378 NEUT (%) 19.20 ± 2.744 18.78 ± 2.977 18.28 ± 1.441 25.50 ± 8.570 LYMPH (%) 73.03 ± 3.627 74.25 ± 3.442 75.13 ± 1.589 67.33 ± 9.64 Plate 3: Rat liver showing slight lymphocyte hyperplasia. Mag (X 250) after 28days treatment with 500mg/kg of the methanol leaf extract of D. saxatilis Plate 4: Rat liver showing slight peri-vascular necrosis Mag (X 250) after 28days treatment with 1000mg/kg of the methanol leaf extract of D. saxatilis ‘b and c’ in superscript represent P< 0.01 and P< 0.001 level of significance respectively. Values are ± SEM, n= 5. Plate 5: Rat kidney showing normal tubules and glomerulus. Mag (X 250) after 28days treatment with distilled water 10 ml/kg Plate 6: Rat kidney showing massive glomerular necrosis with tubular distortion and lymphocyte hyperplasia. Mag (X 250) after 28days treatment with 250mg/kg of the methanol leaf extract of D. saxatilis Table 4: Effect of the methanol leaf extract of D. saxatilis on renal indices Electrolytes Control 250mg/kg 500mg/kg 1000mg/kg Urea (mmol/l) 10.38±1.35 15.46 ± 2.66 14.70 ± 1.42 15.80 ± 2.68 Creatinine(μmol/l) 47.69 ± 6.06 65.05 ± 5.04 59.59 ± 2.59 61.13 ± 4.54 Chloride (mmol/l) 122.60±4.06 105.00 ± 4.56a 107.00 ± 4.64a 111.75 ± 2.96 Sodium (mmol/l) 168.80±7.74 139.00 ± 7.15a 141.20 ± 5.98a 146.00 ± 4.51 Potassium(mmol/l) 5.56±0.27 5.95 ± 0.63 5.50 ± 0.32 5.35 ± 0.18 Bicarbonate (mmol/l) 28.80±2.25 18.50 ± 1.56a 20.80 ± 1.63 21.00 ± 1.16 Plate 7: Rat kidney showing glomerular necrosis with slight tubular distortion and lymphocyte hyperplasia. Mag(X 250) after 28days treatment with 500mg/kg of the methanol leaf extract of D. saxatilis Plate 8: Rat kidney showing intense necrosis of the tubules and glomerulus. Mag(X 250) after 28days treatment with 1000mg/kg of the methanol leaf extract of D. saxatilis ‘a’ in superscript represent P< 0.05 level of significance respectively. n= 5. Values are ± SEM, n = 5. Corresponding Author: Fatima Ismail Hassan, Pharm.fatee@yahoo.com, 08034657833