|Year : 2022 | Volume
| Issue : 2 | Page : 145-150
Sub-acute toxicity studies of the hypocotyl extract of Borassus Aethiopum on hepato-renal functions, and haematological indices in wistar rats
Mansur A Ramalan, Adamu B Shuaibu, Umar S Abdussalam, Abdullahi H Yaro
Department of Pharmacology and Therapeutics, Bayero University, Kano, Nigeria
|Date of Submission||07-Feb-2022|
|Date of Decision||25-Aug-2022|
|Date of Acceptance||20-Sep-2022|
|Date of Web Publication||23-Nov-2022|
Mansur A Ramalan
Department of Pharmacology and Therapeutics, Bayero University, Kano
Source of Support: None, Conflict of Interest: None
Context: Borassus aethiopum is a tropical plant found in the most parts of West Africa. The fruits, sap, and hypocotyl are used for nutritional purposes and in traditional herbal medicine for the treatment of various diseases and infertility. Despite its widespread use, there is insufficient data on its safety profile. Aim: This study is aimed to evaluate the safety of the plant on the hematological indices, liver, and kidney function. Materials and Methods: Twenty Adult male Wistar rats were divided into 4 groups of 5 rats each and were orally administered with different doses of the extract (250, 500, and 1000 mg/kg) and 1 ml of distilled water, respectively, for 28 days. The median lethal dose (LD50) was determined using Lorke's method. The hematological indices, liver and kidney functions of the animals were assessed using standard procedures with the aid of an autoanalyzer. Result: The oral median lethal dose (LD50) was determined to be greater than 5000 mg/kg. No mortality was recorded in the rats after 28 days of daily treatment. With sub-chronic administration, no adverse toxicological effect was observed. There was also no significant difference observed in the hematological, liver, and kidney function parameters between the control group and extract-treated groups. Conclusion: The present study shows that methanol hypocotyl extract of Borassus aethiopum is pre-clinically safe in Wistar rats at the doses tested and may not likely produce any toxic effect on prolonged administration.
Keywords: Borassus aethiopum, median lethal dose, safety, toxicity
|How to cite this article:|
Ramalan MA, Shuaibu AB, Abdussalam US, Yaro AH. Sub-acute toxicity studies of the hypocotyl extract of Borassus Aethiopum on hepato-renal functions, and haematological indices in wistar rats. Niger J Basic Clin Sci 2022;19:145-50
|How to cite this URL:|
Ramalan MA, Shuaibu AB, Abdussalam US, Yaro AH. Sub-acute toxicity studies of the hypocotyl extract of Borassus Aethiopum on hepato-renal functions, and haematological indices in wistar rats. Niger J Basic Clin Sci [serial online] 2022 [cited 2023 Feb 5];19:145-50. Available from: https://www.njbcs.net/text.asp?2022/19/2/145/361898
| Introduction|| |
Borassus aethiopum is a bottle-shaped and smooth palm plant that grows in tropical parts of Africa. It is a member of a genus of six species of fan palms native to tropical regions of Africa, Asia, and New Guinea. It is otherwise known as African Fan Palm, Borassus Palm, or African Palmyra Palm, Deleb Palm, Ron palm, and Toddy's palm. The name is given to the plant because of its fan cluster shaped leaves.
The leaves and roots are used for the treatment of headache, impetigo and other skin diseases epilepsy, fungal infections, malaria, and as aphrodisiacs, while the roots are used as a mouthwash and as a remedy against stomach parasites, bronchitis, sore throats, and asthma.
The fruit of the plant is edible and can be eaten dried or cooked. Various delicacies and dishes are prepared from palm fruit. The juice of the pulp has been reported to be rich in vitamins A and C. Young seedlings, the tuberous portion of the first juvenile leaves, and the apical bud and young leaves are all consumed as vegetables. The seeds of the plant at the beginning of the germination gives a hypocotyl axis which is known locally in Northern Nigeria and parts of Cameroun as known as Muruchi. These young germinating shoots or hypocotyls are usually harvested after 7 to 8 weeks of planting and are consumed roasted or boiled in many parts of Africa especially the Sahelian part. It has been reported to contain a good composition of starch, lipids, fiber, and some micronutrients. Antinutritional factors like Oxalates, phytates, and tannins have been reported in the plant.
Although most edible plants used for dietary, culinary, and medicinal purposes are relatively safe and non-toxic to consumers; however, investigation of medicinal plants and their ability to produce toxic and adverse consequences is of immense importance.[12–14]
It is therefore necessary to evaluate and scientifically examine the possible acute and sub-acute toxicities of plants and spices especially those that are consumed in large quantities.
The beneficial health effects and safety of B. aethiopum have been extensively reported previously; however, its effects on organ functions have not been extensively studied. Therefore, this study was undertaken to determine the possible oral toxicity of the methanol hypocotyl extract of B aethiopum in Wistar rats.
| Materials and Method|| |
The protocol for the study was approved by the Ethical Committee of the College of Health Sciences, Bayero University, Kano, Nigeria with reference number (BUK/CHS/ REC/120), dated 19th November, 2019.
Drugs and assay kits
Reagents and chemicals
Analytical grade chemicals and reagents were used for the study. Assay kits for Alanine amino transferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), and Total Bilirubin were procured from Randox Laboratories LTD., the United Kingdom through their agents in Nigeria (Illupeju Medical Laboratory limited, lagos).
The fresh hypocotyl of Borassus aethiopum was harvested from the soil and collected in October 2019 from Gadama village in Kumbotso Local Government Area of Kano State. A sample of the plant was identified using voucher specimen number BUKHAN 276 and authenticated by Dr Yusuf Nuhu, of the Department of Plant Biology, Bayero University, Kano.
The plant hypocotyl was air-dried under shade at room temperature for one week and grounded into powder using an electric blender. The crude plant part was extracted with seventy per cent methanol (70% methanol, 30% water) by cold maceration for 7 days to ensure maximum extraction with occasional stirring. The resultant mixture was filtered with Whatman No 1 filter paper to remove the marc, and the solvent was allowed to evaporate to dryness over a water bath at 50 degrees Celsius (50°C).
Adult male Wistar rats (10–12 weeks of age) weighing 120–150 g were used for the study. They were obtained from the Animal House, Department of Pharmacology and Therapeutics, Bayero University Kano. The rats were maintained in a well-ventilated room fed with standard rodent chow (vital feed) and permitted free access to water. The animals were allowed to acclimatize to laboratory conditions for two weeks. Experimental and animal handling was carried out according to the guide for the care and use of laboratory animals and guidelines of the institutional ethical committee.
Animal grouping and experimental design
The rats were randomized into 4 groups of 5 rats each namely: group I was given distilled water (1 ml/Kg), while groups II, III, and IV were given 250, 500, and 1000 mg/kg body weight respectively, of the extract corresponding to (5, 10, and 20%) of the LD50. The extract was administered orally daily using the metal gavage cannula.
Determination of LD50 of the hypocotyl methanol extract
The method of Lorke was used to determine the Median lethal dose (LD50). This method is divided into two phases. In phase 1, nine Wistar Rats were divided into 3 groups of three animals each and treated with the methanol extract of the plant at doses of 10, 100, and 1000 mg/kg body weight orally and signs of acute toxicity such as tremors, refusal to feed, respiratory changes and death were observed for 24 hours. After the 24 hours period, there were no signs of toxicity or death in the animals. A second phase was carried out where three fresh rats were divided into 3 groups of one rat each. Each rat was orally administered with 1600, 2900, and 5000 mg/kg of the extract and observed for signs of toxicity and/or death. The LD50 was determined by calculating the geometric mean of the lowest dose that caused death in the animals and the highest dose for which the rat survived.
Sub-acute toxicity tests
A total of 20 male rats were randomly divided into four groups (1, 2, 3, 4) of 5 rats each after being allowed to acclimatize for 2 weeks. The animals were weighed weekly for four weeks. Animals in group 1 were administered distilled water (1 ml/kg), while those in groups 2, 3, and 4 were given graded doses of the extract: 250, 500, and 1000 mg/kg, respectively, corresponding to 5, 10, and 20% of the LD50. At the end of the 28 days experiment, the animals were anaesthetized and sacrificed; blood was collected from the jugular vein in EDTA bottles for hematological studies and plain tubes for liver and renal function indices.
Determination hematological indices
Hematological parameters including red blood cell count (RBC), white blood cell count (WBC), lymphocyte count (LYM), mid cell count (MID), granulocyte count (GRAN), hemoglobin concentration (HGB), packed cell volume (PCV), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red cell distribution width (RDW), and platelet concentration (PLT) were determined using an DxH 560 autoloader hematology analyzer (Beckman Coulter Electronics, Luton, Bedfordshire, UK) with standard calibration, according to the manufacturer's instructions. The auto analyzer was accurately programmed for the analysis of red blood cell (RBC) count, total white blood cell (WBC) count, hemoglobins (Hb) and hematocrit (HCT).
Assay for biochemical parameters
Determination liver function parameters
Biochemical analysis of liver function parameters was performed on serum obtained after centrifugation of whole blood (without Anticoagulant) at 2500 rpm for 5 min. Standardized commercial diagnostic kits (Randox by Randox laboratories LTD., United Kingdom) was used for the determination of the following biochemical parameters: Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), Total bilirubin and Conjugated Bilirubin according to the method of Reitman and Frankel.
Determination Kidney function parameters
Serum urea concentration was determined using the method of Tietz as contained in Randox diagnostic kit.
Principle: This method is based on the principle that urea in the serum is hydrolyzed to ammonia in the presence of urease. The ammonia is then measured photometrically by Berthelot's reaction.
The concentration of Serum creatinine was determined using the method of Tietz. Principle: Creatinine in alkaline solution reacts with picrate to form a colored complex.
Serum sodium and potassium levels were determined using the flame photometry (Sherwood dual-channel model 420C, Cambridge, UK) following the manufacturer's guidelines.
The endpoint calorimetric titration method was used to determine the Cl − concentration and serum bicarbonate concentration was measured by the modified method of Forrester.
The data were analyzed using the statistical package for social sciences (SPSS) version 20.0 The data were expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for comparison between groups. Statistical significance was set at a p value of less than 0.05.
| Result|| |
Median lethal dose of the extract
The Median lethal dose (LD50) of the methanol hypocotyl extract of Borassus aethiopum was found to be greater than 5000 mg/kg in both mice and rats using intraperitoneal and oral routes of administration as reported in previous studies.
Effect of the extract on hematological parameters of male Wistar rats
The extract-treated groups showed a higher mean hematocrit concentration; when compared with the control, this difference was not statistically significant (p > 0.05). The mean values of the neutrophils and lymphocytes counts in the treatment groups were also not statistically significantly different from that of the control (p > 0.05) [Table 1].
|Table 1: Effect of Methanol Hypocotyl Extract of Borassus aethiopum on Hematological Parameters of Male Wistar Rats|
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Effect of the extract on liver function parameters of male Wistar rats
In the extract treated groups, the mean Alanine transaminase (ALT) levels were higher compared to the negative control (Distilled water treated). This difference was not statistically significant (p > 0.05). There was also no statistically significant difference in the other liver function parameters (p > 0.05) except for the levels of the Direct Bilirubin at the dose of 250 mg/kg (you can put the p value [Table 2].
|Table 2: Effect of the Extract on Liver Function Parameters of Male Wistar Rats|
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Effect of the extract on kidney function parameters of male Wistar rats
The mean sodium levels in the extract treatment groups at the doses of 500 mg/kg and 1000 mg/kg were higher than that of the control group (distilled water treated). The observed difference was not statistically significant (p > 0.05). Similarly, other kidney function parameters of the extract treated groups did not differ significantly from the control group (p > 0.05) [Table 3].
|Table 3: Effect of the Extract on Kidney Function Parameters of Male Wistar Rats|
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| Discussion|| |
In this study, it was observed that the rats treated daily with methanol hypocotyl extract of Borassus aethiopum for 28 days have not shown any abnormal gross changes in their behavior such as restlessness, convulsion, hyperventilation, or drowsiness compared to the negative (distilled water treated) control group. This may suggest that the plant at the tested doses is not associated with toxicity that will result in changes of the normal activities of the treated rats. Behavioral impairments and cognitive impairments in animals can occur due to the effect of toxic substances in the central nervous system.[21–23] Therefore, lack of noticeable physical signs of toxicity in the treated rats after 28 days indicates that the plant is relatively safe or non toxic compared to some reported poisonous plants.
Evaluation of hematological parameters such as red blood cells, hematocrit, and hemoglobin is of particular importance in the diagnosis of anemia or detection of deleterious effects on erythrocytes. They are also equally important to assay because the hematopoietic system is a major target for some toxic compounds. In this study the extract of B. aethiopum has not induced any significant increase in red blood cells, hematocrit, or hemoglobin levels of the treated rats. This observation may signify that the plant is safe to the hematopoietic system. A similar observation was seen in a previous study. The major functions of the white blood cells and its differentials are to provide immunity and to defend the body against pathogens and toxins., The total white blood cell count determines the body's ability to fight infection. High white blood cell count is seen when there is acute infection, inflammation, trauma, and cancers such as leukemia, while low white blood cell counts may be caused by problems with their production, auto immune disease where the body fights its cells by mistake or after viral infections. Lymphocytes are the second most common leucocytes circulating in the blood., They are formed in the thymus, lymph nodes, and bone marrow and are essential to the immune defense system as their primary function is to respond to antigens by initiating the immune response. Packed cell volume (PCV) can be used as a screening tool for anemia and can also indicate the degree of fluid loss during dehydration. A drop in packed cell volume (PCV) can indicate internal hemorrhage before any other symptoms become apparent.,,
Many drugs, toxins, and herbal medicines have been reported to cause liver injury and drugs account for 20–40% of all instances of hepatic failure. The activities of amino transaminases (AST, ALT) in the serum are useful indicators of liver damage and are useful in the diagnosis and study of acute hepatic disease.,, Damage to the structural integrity of the liver is reflected by an increase in the serum levels of transaminases because these are cytoplasmic in location and are released into the circulation after cellular damage.,,, In the index study, treatment of the animals with the extract did not cause any significant difference in the concentration of the liver enzymes and serum proteins. Nwachi, 2012 has reported a similar finding. These manifestations may be an indication that there are no adverse metabolic consequences associated with the ingestion of the extract for 28 days.
The kidneys are vital organs that help to maintain the volume, composition, and acid base balance of the total fluid. Many plants, xenobiotics, and drugs interfere these functions. In the clinical laboratory, kidney function tests are used in the assessment of renal disease, water balance, and acid base disorders. The serum levels of Urea, Creatinine, and Electrolytes (Sodium, potassium, chloride, and bicarbonate) are markers of kidney function. The results of the plasma level of Urea, Creatinine, and Electrolytes (sodium, potassium, chloride, and bicarbonate) observed in the study are the indicators of normal kidney function and may imply that the extract is not likely nephrotoxic. This finding is corroborated by previous reports by other scholars.,
| Conclusion|| |
From the findings of this study, it is evident that there was no mortality recorded in the acute toxicity test indicating that the extract of B. aethiopum hypocotyls is relatively non-toxic. The study also shows that the plant extract did not alter the values of the hematological, liver, and kidney functions parameters of male Wistar rats at the tested doses. The plant is practically non-toxic to the Wistar rat at the doses investigated.
The authors acknowledged the kind contributions of the staff of Pharmacology and Therapeutics laboratory, Bayero University, Kano, during the course of this work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]