EVALUATION OF SUB-ACUTE TOXICITY OF THE HYDRO-METHANOL STEM BARK EXTRACT OF BURKEA AFRICANA IN ALBINO RATS

Objective: This study was designed to investigate the sub-acute toxicity profile of hydro-methanol extract of Burkea africana  (BA) stem bark in rats. 
Methods: The stem bark of BA was extracted by cold maceration using 80% methanol. Twenty female albino rats were randomly assigned into four groups of five rats each. Group 1 (only distilled water). Groups 2-4 received the extract (100, 200, and 400 mg/kg) orally, once daily for 28 days. The rats were observed for signs of toxicity and the bodyweight (b.wt) of rats taken weekly. Blood samples were collected on day 28 for hematology and serum chemistry. Visceral organs were harvested for organ-somatic index and histopathology. 
Results: There were no toxicity signs observed and no significant (p< 0.05) change in body weight but the pulmo-somatic index was significantly (p< 0.05) higher at 400 mg/kg compared with the control and other treated groups. Significant (p< 0.05) increase in PCV, RBC, and MCV and significant (p< 0.05) decrease in MCHC, Total WBC count, neutrophils and lymphocytes were observed. Also, there were significant (p< 0.05) decreases in ALT, total protein, globulin, total bilirubin of test groups when compared with the control group. Urea concentration of test groups significantly (p< 0.05) increased when compared with that of the control group. 
Conclusions: BA stem bark extract can be said to have no deleterious effect on erythrocyte, but rather serve to improve erythropoiesis and also has no overt toxic effect on the visceral organs. Also the extract may have immunosuppressive and oxidative tendencies on prolong use. 
                                  
Peer Review History: 
Received 12 January 2021; Revised 3 February; Accepted 25 February, Available online 15 March 2021 
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Received file:                Reviewer's Comments: 
Average Peer review marks at initial stage: 5.0/10 
Average Peer review marks at publication stage: 8.0/10 
Reviewer(s) detail: 
Idoko Alexander, 
Caritas University, Enugu, Nigeria, idokoalexander1@gmail.com 
Taha A.I. El Bassossy,  
Medicinal and Aromatic Plants Department, Desert Research Center, Cairo, Egypt, tahachemist2008@gmail.com 
  
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INTRODUCTION
The act of using plants for treatment, prevention, and control of various disease conditions is an ancient phenomenon 1,2 . Developed countries also have experienced significant increase in the use of herbal remedies, with the belief that they are more efficacious and less harmful 2, 3 . Nonetheless, the fact that they are natural does not make them safe, because little knowledge is available on the safety to validate the claim by manufacturers or traditional healers 2,3 . Many herbal products or medicinal plants have been demonstrated by researchers to be toxic, mutagenic, and carcinogenic 4 . Research has also shown that many medical plants used as herbal remedies contain phytochemical constituents with ability to cause deleterious effect to the body. Such toxic principles include pyrrolizidine alkaloids, benzophenanthrine alkaloids, lectins, saponins, diterpenes, cyanogenicglycosides, and furanocoumarins 2 . Evaluation of medicinal plant and herbal products to determine the level of toxicity in order to establish consequences of long term use is therefore imperative. Toxicity may be seen physically and clinically. Animals or humans may display signs such as restlessness, ataxia, circling, anorexia and subsequently death. The effect of the toxic agent is often seen on the organs especially the liver, kidney, lungs, and heart. This often displays as changes in some biochemical parameters which gives a picture of which organ is mostly affected. Burkea africana (Caesalpiniaceae), a medium size deciduous tree with a wide spread top common in Nigeria is widely used as a remedy for a wide range of ailments in traditional medicine. It has been used often as an anti-venomous agent, cutaneous and subcutaneous parasitic infections, anticonvulsant, hepatic disorders, analgesic, anti-inflammation, antidiarrheal, wound healing, and toothache 5-7 . Empirical evidences exist on its antibacterial, antifungal, larvicidal, molluscicidal, and antioxidant activities 8- 10 . Also, claims for its antidiarrheal, anticonvulsant, analgesic, and anti-inflammatory properties have been reported 7,11, 12 . In this study, the GC-MS analysis and the sub-acute toxicity of methanol stem bark extract of BA were investigated.

Plant material
Fresh stem bark of Burkea africana (BA) were obtained from Ajaba village, a sub-urb of Makurdi metropolis in Benue State and identified by Plant Taxonomists Mr. Yeke Titus of the Department of Forestry, Federal University of Agriculture Makurdi and a voucher specimen number; UAM/FH/0326 assigned and kept in the Departmental Herbarium.

Preparation of plant extract
The BA stem bark was dried in an open shade at room temperature and pounded into smaller piece using a mortar and pestle. This was further made into powdered form using a grinding machine. The powdered material (1000 g) was soaked in 4 L of 80% methanol for 48 h with periodic shaking. The extract was then filtered with a Whatman (No. 1) filter paper. The filtrate was concentrated in a vacuum using a hot air oven at 37 o C into a semi-solid form, yielding in a ratio of 1:10 w/v crude to extract and stored at 4 o C in the refrigerator for future use.

Experimental animals
Female rats weighing 110-120 g were purchased from a commercial animal farm in Nsukka, Enugu State. The animals were kept for seven days in Aluminum cages to acclimatize at the animal house of the Department of Veterinary Physiology and Pharmacology, Faculty of Veterinary Medicine, University of Nigeria Nsukka. During this period, they were provided with potable drinking water and fed adequately with commercially prepared poultry feed pellets (Topfeeds®).The Ethical Committee of the Department of Veterinary Physiology and Pharmacology, University of Nigeria Nsukka gave approval for this research to be conducted with the approval reference number: FVM-VPP-UNN-IACUC-2018-039. The handling and management of animals during this period was in line with good laboratory animal practice regulations as well as the principles of laboratory animal use and care as enshrined by the Natural Research Council guidelines of 2011 13 .

Phytochemical Screening of extracts
The phytochemical screening of the 80% methanol extract of BA stem bark was carried out using standard procedures as described by Trease and Evans 14 and Sofowora 16, 17 . The powdered hydro-methanol extract of BA stem bark was reconstituted to obtain the test aliquot by dissolving 1 g in 500 ml of distilled water. The aliquot was thereafter screened for the presence of alkaloids, flavonoids, tannins, phlebotannins, saponins, glycosides, phenols, terpenoids, steroids, reducing sugar, resins, and volatile oils. Gas chromatography mass spectroscopy One gram (1g) of the methanol stem bark extract of BA was sent to Ahmadu Bello University, Zaria for Gas Chromatography Mass Spectroscopy (GC-MS) analysis (Perkin Elmer Auto sampler XLGC coupled with Turbo Mass Spectrophotometer, Norwalk CTO6859, USA) using analytical conditions described by Adeyemi et al., 17 . The setup had an electron ionization of 70v and the source of the ion had a temperature of 250 o C. Helium gas (99.9% purity) was the carrier gas used, while HP-5ms (30mm X 0.25mm X 0.320µm) was the stationary phase. The oven had a temperature of 80 o C and was kept at that for 5 minutes and then increased to 250 o C. The retention time was 16 minutes, running at the speed of 4 degrees/minute, 1μl was automatically injected to finalize the running time of 50 minutes. Mass Hunter Data Analysis Software was used to analyze and interpret the GC-MS result. Sub-acute toxicity experiment Twenty female albino rats randomly were assigned into four groups. Groups 2-4 were administered the extract at the dose of 100, 200, and 400 mg/kg b.wt respectively, orally for 28 consecutive days. Whereas group 1 served as a negative control and were administered distilled water at 10 ml/kg body weight for the period. The body weights of rats in each group were obtained weekly and recorded accordingly. Blood samples for hematology and serum biochemistry were collected at day 28 post treatments using standard methods. All rats in each group were sacrificed humanely at day 28 and visceral organs (liver, kidney, heart, spleen and lung) were collected, weighed and relative organ versus body weight calculated. Liver and kidneys were preserved using 10% formalin to be used for histopathology examination.

Hematological and serum biochemical analyses
Hematological parameters were evaluated using standard methods 18 . Also, alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) were assayed as described 19,20 . Total serum protein and albumin were evaluated using a clinical refractometer as described by Johnson et al., 21 . Serum globulin concentrations were derived from the difference between total serum protein and albumin. Total and direct bilirubin assay was by the method of Tietz 22 while Urea and creatinine were assayed by the method of Burtis and Ashwood 23 . Also, malondialdehyde, catalase and glutathione were assayed by the methods of Stocks and Dormandy 24 modified by Sicinska et al., 25 , Góth 26 , and Moron et al., 27 .

Histopathological Examination
Tissue samples from the liver and kidney were histologically examined using the conventional staining technique of Hematoxylin and Eosin as described by Drury et al., 28 .

Statistical analysis
All results of this study were expressed descriptively as mean±standard error of mean (S.E.M) and group means were compared using one-way analysis of variance (ANOVA) at significance level of 5% (P< 0.05). Significant differences between means were separated using Duncan multiple range post hoc test. Data was analyzed using SPSS version 21. Bar charts and tables were used to present the data generated in the study.

Sub-acute effects of the extract on organ-somatic index and body weight
Results showed no significant different in the organsomatic index between the control and treated groups for all the organs. However, the pulmo-somatic index was higher in animals treated at the dose rate of 400 mg/kg of the extract as compared with the control and other treated groups ( Figure 1). Also, No significant (P>0.05) difference was observed in the body weights of animals in all the treated groups when compared with the control group ( Figure 2). Table 2 showed significantly (P<0.05) higher values of packed cell volume (PCV) and red blood cells (RBC) in animals treated with the extract at the dose rate of 200 mg/kg and 400 mg/kg as compared to those administered 100 mg/kg dose of the extract as well as the control group.   No significant (P> 0.05) difference in the hemoglobin (Hb) and mean corpuscular hemoglobin (MCH) values between all the treated and the control groups. Also, the mean corpuscular volumes (MCV) were observed to be significantly (P<0.05) higher in animals administered the extract at doses 100 mg/kg and 400 mg/kg compared to the control and the group treated at the dose of 200 mg/kg. Only animals treated with the extract at the dose rate of 100 mg/kg were observed with a significantly (P<0.05) lower MCHC value as compared to both the other two treated and the control groups. Furthermore, result revealed a significantly (P< 0.05) higher total white blood count (TWBC) in those animals treated with 200 and 400 mg/kg dose of the extract as compared with the control group as well as those given a 100 mg/kg dose of the extract. For the neutrophils count, a significantly (P <0.05) lower value was observed in the animals that were treated with the extract at a dose rate of 100 mg/kg when compared with the control and those treated with the higher doses. Again, a significantly (P<0.05) lower lymphocyte count was observed in those animals that were treated with the extract at a dose rate of 400 mg/kg when compared to the control group and the other two groups on lower doses. Furthermore, the result showed no significant (P>0.05) difference between the extract treated and control groups in the observed values of monocytes, eosinophil, and basophils.

Effect on some serum biochemical parameters
In Table 3    The liver of control rats showed normal central venules with the characteristic morphology of the hepatocytes and sinusoids (Plate 1). The kidney tissues of the treated rats were almost same with those of the control. The normal architecture kidney tissue was seen at all doses. The renal cortex also showed normal glomeruli with normal mesengial cells and capsular spaces (Plate 1). The renal tubules, including distal convoluted and proximal convoluted tubules appeared normal with normal interstitial spaces. At the doses 100mg/kg and 200 mg/kg, the interstitial spaces showed areas of mild infiltration of inflammatory cells (Black arrows on Plate 6 and Plate 7).

DISCUSSION
The presence of antioxidants such as phenols and flavonoids, saponins, tannins and terpenoids in the methanol stem bark extract of Burkea africana suggests its anti-oxidative stress potential. There are many reports on the antioxidant, antimicrobial, antinflammatory, anti-angionic, analgesic, anti-allergic, cytostatic and properties of these phytochemicals suggesting wide range of biological activities 7,12 . The GC-MS result revealed the presence of (2H) pyrrole-2carbonitrile, 5-amino-3, 4-dihydro-, an alkaloid and 9, 17-octadecadienal, (Z)-, an unsaturated aldehyde which has been found to have antimicrobial and antiinflammatory activities 29, 17 . This probably explains the findings of Tor-anyiin and Anyam 12 , and Musa et al., 7 . Resorcinol, a phenolic compound is a known antioxidant with hepatoprotective activity 9 This corroborates a report by Cordier et al., 9 that the plant is rich in phenol, making it a potent antioxidant. Oleic acid which is also a fatty acid has been proven to be a potent antihypertensive and is found to be in abundance in olive oil 30  antifungal, larvicidal, molluscicidal 12 and antiinfluenza 10 activities of this plant. Knowledge of the possible toxic or adverse effects of many medicinal plants is grossly inadequate. In evaluating the safety status of medicinal plant, acute, sub acute and sometimes chronic toxicity studies are carried out in laboratory animals 2 . In this study, daily oral administration of methanol extract of Burkea africana stem bark at the doses of 100, 200 and 400mg/kg body weight for 28 consecutive days did not cause any change in behavior or mortality in treated rats, suggesting that the extract is relatively safe. Sign of toxicity such as sedation, lethargy, anorexia, drowsiness and ultimately death have been used to evaluate toxic effect of chemicals and natural medicinal plant products used in traditional medicine by scientists. The absence of these signs is used as a criterion to support that the plant extract is safe for use medicinally 32 . There was no significant (P <0.05) effect on the body weight and organ-somatic index of the treated rats compared with the normal control ( Figure 1 and Figure 2). These findings indicate that the extract showed no adverse effect on the organs (liver, kidney, lungs, heart and spleen) at all doses used in this study and therefore is considered to be safe. Also, the extract maybe said to have no anti-nutritive and growth inhibiting effect since it had no effect on the body weight. According to Unuofin et al., 2 , weight loss of about 10% has been related to an adverse effect.
In the same vain, organ-somatic index is often used in toxicological investigations 33-35 . After 28 days of a single daily oral administration of methanol extract of Burkea africana stem bark, hematological parameters showed some significant changes ( Table 2). The fact that the hematopoietic system is readily attacked by toxic substances makes it imperative to always evaluate hematological parameters in toxicity studies to monitor the physiologic and pathological state of animals and humans 2 . The PCV and RBC count of treated groups increased significantly in a dose-dependent manner, with no significant effect on hemoglobin. Hemoglobin, MCH and MCHC remained unaffected, with significant (p<0.05) increase in MCV of treated groups at all doses. Circulating blood carries oxygen, nutrients and foreign substances, making it prone to toxic attacks leading to damages in RBCs, WBCs, platelets and hemoglobin. This gives rise to various forms of anemia depending on the component of the RBC affected and nature of the effect and also immune system failure 2 .
The results of this study suggests that the extract probably has stimulatory effect on erythropoiesis and hence useful in the treatment of anemia. The decreases in Leucocytes at 200 and 400 mg/kg, neutrophils at 100 mg/kg, and lymphocytes at 400 mg/kg body weight observed could be due to immunosuppressive potential of the extract. These changes may also be due to inflammatory response and/or stress 36 . The effect of methanol extract of Burkea africana stem bark on the liver was assessed by evaluating serum activities of liver enzymes. The enzymes (AST and ALT) activities are often used to evaluate the functional status of the liver and the condition of the hepatocytes due to the high amount of these transaminases found in the hepatocytes 37,38 . However, ALT is considered more specific to liver 37-39 . Treatments with this extract significantly decreased (P<0.05) the serum activities of ALT after 28 days of oral administration, with no effect on AST and ALP. This suggests the absence of hazardous effect of the extract on the liver. The decrease in serum total protein observed could be due to decrease in globulin. This may be thought to be from the effect of some components of the extract on lymphoid organs with possibility of liver involvement 37, 40 . Albumin increased significantly when rats were treated with extract at 100 and 200 mg/kg. Studies haves shown that albumin concentration and function in liver cirrhosis is often reduced 41 , which further corroborate with our earlier suggestion that the extract has no adverse effect on the liver. The extract at 100 mg/kg slightly increased total bilirubin, whereas direct (conjugated) and indirect (unconjugated) bilirubin remained unaffected ( Table 5), suggesting that there is no problem with bilirubin conjugation in the liver. Hemoglobin metabolism which takes place in the liver, spleen and bone is the major source of bilirubin in the serum 3 . Elevated serum bilirubin is due to increased destruction of erythrocytes resulting to increased release of hemoglobin as well as obstructive liver disorders 34, 37 . The extract may be said to have bile ducts obstructing tendencies, which is one of the major causes of increased serum bilirubin 37 . This also is in doubt considering the fact that ALP was consistently unaltered throughout the period of treatment at all doses used in this study. Urea and creatinine are used to evaluate the functional status of the kidney, although serum creatinine concentration is considered a more reliable marker for evaluation of kidney function 37, 42 . The kidney as an excretory organ, is prone to toxic attack. This toxic effect on the kidney often result in impaired renal functions such as impaired excretion of metabolic waste, maintenance of fluid and electrolyte balance, and hormonal imbalance due to impaired synthesis of such hormones (erythropoietin) 3 . Serum urea and creatinine concentrations increase due to inability of the kidney to excrete urea and creatinine proportionately to their formation 3,37 . Daily treatment with methanol stem bark extract of B. africana showed serum urea levels to be elevated at 400mg/kg b. wt. Elevated MDA and decreases in GSH (Table 8) observed is an indication that the crude extract enhances lipid peroxidation and free radical formation when administered for a long period 37 . Malondialdehye (MDA) is end result of lipid peroxidation due to increased free radical production or decrease in antioxidant defense system 43-45 . Reduced glutathione is a natural antioxidant in the liver and serves to conjugate with toxic metabolite, making them more polar and readily excreted 45 . Glutathione also serves to scavenge free radicals and reduce oxidative effect in cells and eventual cell death. Therefore the ability of cells to sustain GSH concentration is useful for cell function and survival 46 . Cereser et al., 46 postulated that low GSH with corresponding decrease in glutathione reductase enzyme (GR) creates an oxidative imbalance, inspiring oxidative processes and then cell death. Reduction in GSH is marked by increase lipid peroxidation caused by free radical reaction seen as increased MDA 46 . The significant (P<0.05) decrease in GSH in the treated group at the doses used in this study suggest that the extract may have inhibitory effect on the enzyme glutathione reductase which reduces oxidized glutathione (GSSH) thereby depleting reduced Glutathione (GSH). This explains the increased MDA observed in this study. Decrease in catalase activity in serum can be due to imbalance in its utilization and synthesis or as a problem with expression in the gene controlling its synthesis, resulting in oxidative stress and tissue damage induced by precursors of oxidation (pro-oxidants) 9 . In the subacute administration of the extract; catalase was significantly elevated compared to the normal control. This is an indication that the extract has some stimulatory effect on catalase activity and release which further explains the antioxidant properties of this plant in spite of the increased lipid peroxidation and decrease in GSH. Catalase is a very potent antioxidant enzyme in cells and a molecule of catalase can neutralize millions of peroxide molecules to water and oxygen in seconds 47 . Histopathology of the liver and kidney revealed little or no pathological effect that is due to the treatment with methanol extract of Burkea africana stem bark. Pathological changes in the parenchymal cells of the liver are often associated with changes in serum activities of liver enzymes 48 . Absence of necrosis of the hepatocyte in the treated groups further agrees with the results of the enzyme assay ( Table 3). The mild infiltration of inflammatory cells noticed at the periportal region and in the sinusoid is to be considered none pathologic. The kidney cellular morphology appeared normal, indicating that its functional status may not have been altered by the extract. This is further substantiated by the serum urea and creatinine levels ( Table 3).
In conclusion, the methanol extract of Burkea africana stem bark administration orally for consecutive 28 days up to a dose of 400 mg/kg body weight had no obvious deleterious effect in rats. The results in this study suggest that the plant is safe for use in treatment of the claimed ailments and therefore supports its use in traditional medicine by rural dwellers. Therefore the use of this plant in treatment is best if used for short durations or at lower doses.

AUTHOR'S CONTRIBUTION
TF Swem take care of the laboratory work, PE Aba was involved in designing the experiment, interpreted the data. SC Udem conceived the study, contributed in the design of the experiment, VM Ahur and FA Gberindyer interpreted the clinical chemistry and hematology results.