The study was to investigate the safety margins of ethanol extract of
This research is a toxicology investigation.
The acute and sub-acute toxicity studies conducted on the EEIGRB, according to the Organization for Economic Cooperation and Development (OECD) methods.
The acute toxicity studies revealed that LD50 was > 5000 mg/kg. In the sub-acute study, significant increase in body weights (
The results indicate that EEIGRB was found to be practically safe after acute administration, and there were histomorphological alterations in the liver and kidney after prolonged administration in the sub-acute dosages.
The practice of traditional medicine and the use of medicinal plants in most developing countries as a normative basis for the maintenance of good health have been widely reported (Jordan, Cunningham & Marles
Two kilograms of powdered root bark of
The EEIGRB was subjected to qualitative phytochemical tests as described by Evans (
Wistar rats of both sexes weighting 100 – 140 grams were sourced from the Animal Facility Units of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria. They were managed in well-ventilated cages at room temperature under normal day and night cycle, kept on pelletised animal feed (Vital feed®, Jos) with access to water
Acute oral toxicity-limit dose method was adopted by the Organization for Economic Cooperation and Development (OECD Test Guideline 425). Six rats were used; two rats were picked, weighed and given EEIGRB orally with a dose of 2000 and 5000 mg/kg body weight. The rats were observed for 30 min, 4 h and the 24 h for signs or symptoms of toxicity, before introducing EEIGRB to the remaining four rats. The observation included: changes in skin and fur, eyes and mucous membrane and respiratory and behaviour pattern. Animals were observed for signs and symptoms of toxicity and mortality for 14 days (OECD
The study was carried out following OECD (
The body weights of the animals were recorded at the beginning of the experiment and repeated at a 7-day interval until the termination of the experiment. Doses of EEIGRB administered were adjusted accordingly. On the 29th day, the rats were weighed; blood was pricked from the tail for haematological evaluation, before sacrificed. The animals’ anatomical sections were dissected carefully to obtain the kidney, liver, heart, spleen and lungs. All organs were weighed and observed macroscopically. The relative organ–body weight (ROW) ratio of each rat was calculated as follows:
The blood samples for the haematological tests were collected into vacuum tubes containing ethylene diamine tetra-acetate acid (EDTA) as anticoagulant and taken to the laboratory. Blood indices including white blood cells (WBCs), red blood cells (RBCs), haemoglobin (HB), platelets, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were analysed by using an automated haematology analyser (Cell-DynTM Abbott, United States [US]).
The blood samples for biochemical analyses were collected into plain universal bottles, allowed to clot and centrifuged at 3000 revolutions per minute (rpm) for 10 min. The serums obtained were analysed to estimate the effect of EEIGRB on biochemical indices by using a photoelectric colourimeter (AC-115 Optima, Japan). The enzymes parameters estimated were alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein, albumin, creatinine, urea and electrolytes (chloride, sodium, potassium and bicarbonate ions) as liver and kidney function tests performed by colourimetric method by using Randox assay kits and automated biochemistry analyser (Jaijoy et al.
The kidney and liver of the sacrificed rats were harvested and cut into 3-cm-thick slices and fixed in 10% formalin solution for sectioning. The fixed specimens were sliced, processed and embedded into paraffin blocks. The blocks were cut into 5-micrometre (µm)-thick, paraffin sections by a rotary microtome. The sections were stained with haematoxylin and eosin H and E for histological observations. The stained sections were finally viewed under a light microscope for morphological changes (Rolls
Data obtained from the study were analysed by using Statistical Package for Social Sciences (SPSS), IBM version 20. Descriptive statistics were carried out to obtain the mean ± standard error of mean (SEM). Data on ROW and haematological, hepatic and renal indices were analysed by one-way analysis of variance (ANOVA), whereas those on body weight were analysed by using repeated-measures ANOVA and Bonferroni test for comparison over time. Statistically significant differences were considered at 95% and 99% confidence intervals (
The animals were treated following the standard guidelines for the Care and Use of Laboratory Animals and with an approval of the Animal Ethics and Care Committee, Ahmadu Bello University, Nigeria (ABUCAUC/2018/096).
The phytochemical analysis of EEIGRB revealed the presence of saponins, anthraquinones, cardiac glycosides, flavonoids, tannins, alkaloids, steroids and triterpenes.
The limit test results revealed that a single oral dose of EEIGRB did not cause mortality and signs of toxicity to the rats within 24 h and during the 14 days of observation period. It showed that the LD50 was > 5000 mg/kg body weight in rats.
The oral administration of EEIGRB at doses of 100, 200 and 400 mg/kg body weight produced no observable obvious signs and symptoms of toxicity such as tiredness, weakness, convulsion, hyper-activeness, dullness, diarrhoea and diuresis throughout the 28 days of study period. All the animals survived throughout the experiment.
There was a progressive increase in the weight of the rats in all the groups over the 28 days. However, there was a significant increase in body weights at weeks 1, 2, 3 and 4 compared with week 0 in the control group at
Effect of 28 days of oral administration of ethanol extract of
The 28 days of oral administration of EEIGRB showed no significant (
Effect of 28 days of oral administration of ethanol extract of
Oral administration of EEIGRB produced no significant (
Effect of 28 days of oral administration of ethanol extract of
Haematological indices | Treatment groups |
|||
---|---|---|---|---|
D/W (1 mL/kg) | EEIGRB 100 mg/kg | EEIGRB 200 mg/kg | EEIGRB 400 mg/kg | |
WBC (×109/L) | 4.83 ± 0.12 | 4.77 ± 0.49 | 4.63 ± 0.38 | 5.13 ± 0.41 |
RBC (×109L) | 6.00 ± 0.10 | 6.07 ± 0.12 | 6.00 ± 0.05 | 6.07 ± 0.12 |
HB (g/dL) | 12.43 ± 0.23 | 13.23 ± 0.82 | 12.50 ± 0.44 | 13.80 ± 0.44 |
PCV (%) | 37.67 ± 1.33 | 40.33 ± 2.60 | 37.00 ± 1.00 | 42.33 ± 1.45 |
MCV (fL) | 88.40 ± 1.00 | 83.77 ± 2.53 | 88.13 ± 0.69 | 87.53 ± 0.03 |
MCH (pg) | 29.53 ± 0.62 | 28.43 ± 1.38 | 29.87 ± 0.45 | 29.73 ± 0.55 |
MCHC (g/dL) | 33.40 ± 0.57 | 33.47 ± 0.17 | 31.90 ± 1.15 | 31.67 ± 0.24 |
Platelets (×109L) | 226.33 ±40.99 | 191.00 ± 18.00 | 179.33 ± 5.78 | 175.67 ± 16.18 |
LYMPH% | 60.30 ± 1.76 | 60.20 ± 3.04 | 55.23 ± 1.78 | 59.57 ± 1.37 |
GRAN% | 35.27 ± 1.87 | 35.33 ± 1.82 | 39.70 ± 1.39 | 35.53 ± 1.83 |
MID% | 4.73 ± 0.03 | 5.97 ± 0.49 | 5.10 ± 0.50 | 4.40 ± 0.75 |
Note: Values are expressed as mean ± standard error of mean. There were no significant differences between values for control and treatments groups (distilled water).
D/W, distilled water; EEIGRB, ethanol extract of
The 28 days of oral administration of EEIGRB did not produce significant (
Effect of 28 days of oral administration of ethanol extract of
Liver biomarkers | Treatment groups |
|||
---|---|---|---|---|
D/W (1 mL/kg) | EEIGRB 100 mg/kg | EEIGRB 200 mg/kg | EEIGRB 400 mg/kg | |
ALT (IU/L) | 40.75 ± 1.03 | 38.00 ± 1.00 | 38.67 ± 2.72 | 47.67 ± 8.65 |
AST(IU/L) | 242.50 ± 4.05 | 235.33 ± 7.69 | 241.00 ± 6.08 | 243.33 ± 6.77 |
ALP (IU/L) | 35.78 ± 2.18 | 33.70 ± 4.16 | 30.80 ± 0.85 | 43.50 ± 3.36 |
TP (g/dL) | 12.13 ± 0.54 | 10.87 ± 0.44 | 11.80 ± 1.03 | 12.50 ± 1.06 |
Albumin (g/dL) | 2.93 ± 0.10 | 2.67 ± 0.09 | 3.00 ± 0.17 | 2.97 ± 0.17 |
Note: Values are expressed as mean ± standard error of mean.
ALB, albumin; ALP; alkaline phosphatase; ALT, alanine aminotransferase; AST; aspartate aminotransferase; D/W, distilled water; EEIGRB, ethanol extract of
, The mean difference is statistically significant (
A significant reduction in serum concentrations of sodium and creatinine at
Effect of 28 days of oral administration of ethanol extract of
Kidney biomarkers | Treatment groups |
|||
---|---|---|---|---|
D/W (1 mL/kg) | EEIGRB 100 mg/kg | EEIGRB 200 mg/kg | EEIGRB 400 mg/kg | |
Urea ( |
31.25 ± 2.99 | 34.43 ± 2.41 | 29.47 ± 1.47 | 37.77 ± 3.99 |
Creatinine ( |
1.03 ± 0.08 | 0.79 ± 0.06 | 0.73 ± 0.12 | 0.67 ± 0.03 |
Na+ (mmol/L) | 169.13 ± 3.57 | 171.47 ± 1.73 | 165.57 ± 3.83 | 145.30 ± 3.00 |
K+ (mmol/L) | 21.88 ± 1.46 | 20.00 ± 4.39 | 17.43 ± 1.71 | 20.37 ± 1.39 |
Cl− (mmol/L) | 108.25 ± 6.80 | 105.00 ± 5.77 | 91.00 ± 2.00 | 91.33 ± 1.86 |
HCO3− (mmol/L) | 24.00 ± 3.67 | 31.00 ± 1.15 | 30.00 ± 5.51 | 25.00 ± 1.73 |
Note: Values are expressed as mean ± standard error of mean.
Cl−, chloride ion; D/W, distilled water; EEIGRB, ethanol extract of
, The mean difference is statistically significant (
Histological examination of the liver revealed a vascular congestion at a dose of 100 mg/kg and a slight hepatic necrosis at 200 mg/kg of EEIGRB. Moderate foci necrosis and lymphocyte hyperplasia were observed at 400 mg/kg of EEIGRB (
Photomicrograph of the liver section of (a) control group rats (1 mL/kg distilled water) showing normal hepatocytes; (b) after a dose of 100 mg/kg of ethanol extract of
Photomicrograph of the kidney section of (a) control group rats (1 mL/kg distilled water) showing the normal glomerulus and tubules; (b) after a dose of 100 mg/kg of ethanol extract of
Preliminary phytochemical screening gave a brief idea about the qualitative nature of active phytochemical constituents present in plant extracts, which will help the investigators in future regarding the selection of the particular extract for further investigation or isolating the active constituent(s) (Mishra et al.
The body weight changes serve as a sensitive indicator of the general health status of animals. Changes in body weight have been used as an indicator of adverse effects of drugs and chemicals (Nandy & Datta
Organ weight is also an important index of the physiological and pathological status in animals. The relative organ weight is a more viable and sensitive index of toxicity than absolute organ weight as it relates the overall well-being of the animals to each of their organs (Joth et al.
The haematopoietic system is a susceptible target for toxic compounds and an important index of physiological and pathological status in man and animals, especially in the bone marrow where the production of RBCs occurs (Kifayatullah et al.
Liver function tests involve evaluating serum ALT, AST, ALP, bilirubin and albumin levels. The most commonly used indicators of liver injury are the ALT and AST in the blood stream or plasma, which usually suggests chronic hepatitis or biliary obstructions (Ramaiah
Creatinine and urea are considered as important prognostic markers of renal dysfunction and kidney failure for any toxic compound (Sood et al.
Histopathological examination is one of the gold standards for evaluating treatment-related pathological changes in tissues and organs (OECD
The EEIGRB was found to be relatively safe after acute administration and showed mild toxicity after 28 days of repeated oral administration. However, EEIGRB was found to have hepatic toxicity and nephrotoxicity with associated histomorphological alterations in the liver and kidneys at an oral dose of 400 mg/kg for 28 days; therefore, prolonged oral administration of EEIGRB should be avoided because of the toxicity risk.
The authors thankfully acknowledge the technical support of the entire staff of the Animal Facility Units of the Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Science, Ahmadu Bello University, Zaria, Nigeria, during the course of this work.
The authors have declared that no competing interests exist.
A.N. designed and conducted the experiment and drafted the manuscript. E.M.A., U.H.D. and M.U.K. designed the experiment, gave directions and proof-read the manuscript. A.M.Z. and A.E.A. participated in the data analysis, edited and proof-read the manuscript..
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Data sharing is not applicable to this article as no new data were created or analysed in this study.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.