Toxicity Assessment and Anxiolytic Properties of Methanol Stem Bark Extract from Khaya senegalensis in Wistar Rats

Background and Objective: Mental illnesses such as anxiety are characterized by impairment in cognition, emotional regulation, or behavior and are predisposed by a combination of genetic, biological, environmental and psychological factors. Khaya senegalensis is reported to have a wide variety of medicinal uses such as in diarrhea, bacterial infections, helminthiasis, trypanosomiasis, diabetes and cancer. This study evaluated the anxiolytic effect of methanol stem bark extract of Khaya senegalensis in Wistar rats. Materials and Methods : Acute toxicity studies were conducted using Lorke’s Method. After which thirty rats were randomly divided into five groups of six each, group 1 and 5 received distilled water and diazepam, respectively as control groups and 2nd to 4th group received graded doses of methanol stem bark extract of Khaya senegalensis for anxiety studies using Hole-board Test and Elevated Plus-Maze. Results: The extract significantly (p<0.05) increased the number of entries into open arms and the time spent there in the Elevated Plus-Maze Test and increased the number of head dips in the Hole-board Test. Conclusion: This study revealed that the methanol stem bark extract of khaya senegalensis is practically nontoxic and also demonstrated significant and dose-dependent anxiolytic activities in Wistar rats.


INTRODUCTION
The history of using plants as a source of medicine is as old as humankind 1 . There has been increasing interest in the utilization of herbal medicines over the last few decades. This is because medicinal plants are useful in treating so many diseases, including mental illnesses such as anxiety and depression.
Mental illness refers to a wide range of conditions characterized by abnormalities of mood, thinking and behavior 2 . Worldwide, mental illness accounts for 14% of the global disease burden with 75% of the Extract preparation: The stem bark was dried under shade then chopped into small pieces and pulverized into a fine powder using a wooden pestle and mortar. Five hundred grams of the dried powder was subjected to cold maceration using 95% methanol as solvent, allowed for 72 hrs and filtered using Whatman filter paper. The filtrate was evaporated in an oven at 40°C. The dried extract was stored in an air-tight container. The percentage yield was calculated by dividing the total weight of the dried extract by the total weight of the dried powdered plant, then multiplying the value by 100.
Acute toxicity studies: Lorke's Method was adopted for this study 18 . The test was conducted in two phases. In the initial phase, nine rats were divided into three groups with three rats each. First, second and third groups received 10, 100 and 1000 mg kgG 1 of Khaya senegalensis stem bark extract, respectively and were observed for 24 hrs for possible mortality. In the second phase, three rats, one from each group used and received 1600, 2900 and 5000 mg kgG 1 of the plant extract respectively. The LD50 was determined by calculating the geometric mean of the highest dose the rat survived and the lowest dose that killed the rat.

Anxiolytic test
Elevated Plus-Maze Model: Lister's Method was adopted for this study 19 . The apparatus comprised two open arms and two closed arms extending from a common central platform. The maze was entirely elevated to a height of 50 cm from the ground. Thirty rats were grouped into five groups with six each being used for the experiment. Group I was labelled positive control, given 1 mg kgG 1 of diazepam orally and allowed 30 min for the drug to act. The rat was then put into the maze and observed for 3 min. The subsequent groups of rats were subjected to similar tests after administering 100, 200 and 400 mg kgG 1 of Khaya senegalensis stem bark extract and distilled water via oral route, respectively. The parameters observed include (1) Number of entries into the open arm, (2) Number of entries into the closed arm and 3 times spent in the arms, respectively.
Hole-board Test: The method described by Takeda et al. 20 was adopted. The hole-board consists of a wooden box with sixteen holes evenly distributed on the floor in a grid pattern. The apparatus was elevated to about 50 cm above the ground. Thirty rats were grouped into five groups with six each being used for the experiment. Group I was labelled positive control and 0.5 mg kgG 1 of diazepam was given to each of the rats in the group orally and allowed 30 min for the drug to act. Then each rat was put on the board and observed for 3 min. The subsequent groups of rats were subjected to similar tests after administering 100, 200 and 400 mg kgG 1 of Khaya senegalensis stem bark extract and distilled water via oral route, respectively. The number of head dips was observed and recorded.

Sub-chronic toxicity studies:
The Organization for Economic Co-operation and Development (OECD 407) method was adopted for the study's 28-day sub-chronic toxicity test 21 . Twenty four rats were allocated into four groups of six at random. Orally administered to the first group was 10 mL kgG 1 of distilled water. Statistical analysis: The data were analyzed using SPSS Version 24 and summarized as mean±standard error of mean (SEM). One-way Analysis of Variance (ANOVA) and Mann-Whitney U Test were used to compute the differences. After obtaining statistical differences, the Dunnett's Test was used for multiple comparisons depending on the nature of the data. The p<0.05 was accepted as significant.

Percentage yield of methanol stem bark extract of Khaya senegalensis:
The percentage yield of methanol stem bark extract of K. senegalensis was 9.2%. Acute toxicity studies: No mortality is observed in both phases of the acute toxicity study ( Table 1). The median Lethal Dose (LD 50 ) of methanol stem bark extract of K. senegalensis was calculated to be more than 5000 mg kgG 1 orally.
Effect of methanol stems bark extract of K. senegalensis on liver function test following 28 days' sub-chronic oral treatment in Wistar rats: There was no significant change in all the liver function parameters following 28 days of oral administration of graded doses (100, 200 and 400 mg kgG 1 ) of methanol stem bark extract of K. senegalensis in Wistar rats ( Table 2).

Effects of methanol stem bark extract of K. senegalensis on renal function test following 28 days sub-chronic oral treatment in Wistar rats:
There was no significant change in all the renal function parameters following 28 days of oral administration of graded doses (100, 200 and 400 mg kgG 1 ) of methanol stem bark extract of K. senegalensis in Wistar rats ( Table 3).

Effects of methanol stem bark extract of K. senegalensis on hematological indices following 28 days sub-chronic oral treatment in Wistar rats:
There was no significant change in all the hematological parameters following 28 days' oral administration of graded doses (100, 200 and 400 mg kgG 1 ) of methanol stem bark extract of K. senegalensis in Wistar rats ( Table 4).

Effect of methanol stem bark extract of K. senegalensis on anxiety behavior using Hole-Board Test in Wistar rats:
The oral administration of methanol stem bark extract of K. senegalensis to Wistar rats at a dose of 400 mg kgG 1 showed significant (p<0.05) increase in head dips on hole-board test compared to control, but not as much as the standard anxiolytic (diazepam) at a dose of 1 mg kgG 1 as shown in

Effect of methanol stem bark extract of K. senegalensis on anxiety behavior using Elevated Plus Maze in rats:
Oral administration of methanol stem bark extract of K. senegalensis at a dose of 400 mg kgG 1 , when compared to the control, showed a significant increase (p<0.05) in the number of entries into the open arms and the time spent there (Fig. 2), as well as a reduction in the number of entries into the closed arm (Fig. 3), though not as much as with the conventional anxiolytic (diazepam) at a dose of 1 mg kgG 1 .

DISCUSSION
The oral acute toxicity studies of the methanol stem bark extract of K. senegalensis were estimated to be greater than 5000 mg kgG 1 b.wt. In grading the toxicity of a chemical substance, Lorke 18 , indicated that toxicity at 1 mg kgG 1 is considered highly toxic, 10 mg kgG 1 is considered toxic, 100 mg kgG 1 moderately toxic, 1000 mg kgG 1 slightly toxic and 5000 mg kgG 1 not toxic. Therefore, the extract is practically non-toxic. The LD 50 of the aqueous leaf extract of the same plant was reported to be greater than 3000 mg kgG 1 22 .
This study shows no significant difference in liver enzymes after administration of various doses (100, 200 and 400 mg kgG 1 ) of methanol stem bark extract of K. senegalensis to Wistar rats for 28 days compared to the control group. This was contrary to the findings of Kolawole 23 24 with much lower doses (10-40 mg kgG 1 ) administered for 28 days in albino rats. Yakubu et al. 25 also reported an increased concentration of liver enzymes in rats following daily administration of ethanolic extract of K. senegalensis at 2 mg kgG 1 for 18 days. These authors, therefore, concluded the cytolytic effect of the extract on the liver. However, some authors reported reduced serum AST, ALT and ALP effects after using the extract on rodents and therefore highlighted the possible hepato-protective effect of the extract [26][27][28] . The discrepancy in the above findings could be due to impurities such as heavy metals in the extract, which may be toxic.
The current study showed no significant statistical difference in renal parameters following graded doses (100, 200 and 400 mg kgG 1 ) of methanol stem bark extract of K. senegalensis compared to the control after 28 days. El Badwi et al. 29 revealed the nephro-protective effect of K. senegalensis aqueous extract in Wistar rats with gentamycin-induced renal dysfunction after receiving 250-500 mg kgG 1 b.wt., of the extract, having significant improvement in their renal function. However, Kolawole 23 , reported a dosedependent increase in the blood level of urea, creatinine, total protein and globulin, in rats following 100 and 200 mg kgG 1 administered for 18 days. On the contrary, Ali et al. 26 reported reduced concentration of the exact parameters after using higher doses (250 and 500 mg kgG 1 ) for a shorter duration of five days. The disparity between the two observations suggested that a longer course of treatment can cause histological damage to the kidneys, liver and other organs. Some authors have also reported increased serum sodium and potassium after administering the extract to the rats 23,30 .
This study also has not shown any significant change in hematological parameters. However, Kolawole 23 revealed a decrease in red blood cells, packed cell volume and hemoglobin concentration. This may indicate toxicity to the erythrocytes.
This study showed that methanol stems bark extract of K. senegalensis exhibited anxiolytic activity as demonstrated by Hole-Board Test (HBT) and Elevated-Plus Maze (EPM). Rodents have a natural aversion to new, bright, open and elevated places. The exploratory activity reflects the combined effects of these tendencies in novel situations. The number of head dips is assumed to be inversely proportional to the anxiety state 31 . Therefore, the HBT increase in head dips is suggested as an index of anxiolytic activity. Similarly, the EPM is based on a similar principle: Rodents have a natural tendency to explore the novel environment and their innate avoidance of unprotected (open), bright and elevated places. Confinement to the open arm caused physiological stress which manifests as increased defecation, whereas exposure to classical anxiolytics such as diazepam or extract with anxiolytic properties increased exploration of these arms 32 . Other studies in different parts of the world used similar animal models, especially EPM and HBT, to evaluate the anxiolytic effects of different plants and found similar results [33][34][35] .
The results obtained from the present study using the HBT and EPM models for anxiety, revealed that oral administration of methanol stems bark extract of Khaya senegalensis at a high dose of 400 mg kgG 1 produces a significant increase in the number of head dips in HBT and the number of open arm entries and the time spent there in EPM, this suggested that the extract possess anxiolytic effects.