1. 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
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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< 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< 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 ± ±
225.40±
1.29 ± 0.191
250mg/kg
± a
± a ±
0.94± 0.147
500mg/kg
± 6.909a ± ±
0.99 ± 0.182
1000mg/kg
95.34 ± 6.585 ± ±
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 g 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 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±
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±
2.092 x 10-1± 0.198
1.107x10-2±
2.094 x 10-1± 0.198
Spleen
7.458 x10-3±
2.050 x10-1± 0.199
5.169 x10-3±
2.045 x10-1± 0.198
Kidney
6.939 x10-3±
2.057 x10-1± 0.198
7.287 x10-3±
2.057 x10-1± 0.199
Liver
3.361 x10-2±
2.279 x10-1± 0.193
3.408 x10-2±
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:
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:
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):
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:
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)
± 3.774
± 2.273
± 6.419 ±
WBC(x 109/L)
14.04 ± 1.129
8.08 ± b
6.10 ± c
6.68 ± b
MCHC (g/L)
± 5.206
± 2.496
± 3.755
± 3.881
PLT (x 109)
698.00±63.234
544.00±52.857
597.20±70.532 ±
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< 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
± 4.56a
± 4.64a
± 2.96
Sodium (mmol/l)
168.80±7.74
± 7.15a
± 5.98a
± 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,