Evaluation of the Toxicological Effects of the Ethanolic Extract of Alstonia Boonei Stem Bark

ABSTRACT

This study investigated the toxicological effects of ethanolic extract from alstonia boonei stem bark, based on the dosage and duration of administration to albino mice. We evaluated for the phytochemistry of the stem bark and the clinical biochemical parameters of blood plasma of albino mice. After administering the aqueous extract to the albino mice, the biochemical parameters in their blood plasma were determined using commercially available kits. The determination of the phytochemical constituent of the ethanolic extract revealed the presence of alkaloids, flavonoids, saponins, terpenoids and cardiac glycosides. After the first week of administration, no significant variation in most of the biomarkers was recorded. However, after second week of treatment, urea and alanine aminotransferase levels fell significantly. Consequently, the ratio of aspartate transaminase to alanine transaminase was found to be much greater than 2, which was attributed to alcoholic toxicity from the ethanolic extract consumption.

KEY WORDS: Anti-malarial, alstonia boonei, ethanolic extract, toxicology, albino mice

INTRODUCTION

Malaria is a life-threatening disease caused by a parasitic infection of the red blood cells and transmitted through a bite of the female anopheles mosquitoes (1). It is undoubtedly the single most destructive and dangerous infectious agent in the developing world, predominantly in tropical and subtropical regions, including parts of the Americans, Asia and Africa (1-4). The World Health Organisation and the World Bank rank malaria as the largest single component of the disease burden in Africa, causing an annual loss of 35 million future life-years from disability and premature mortality (5). In Africa, malaria is responsible for about 20-30% of hospital admissions and about 30-50% of outpatient consultation (3).

In recent times there has been an upsurge in the popularity and the use of plant extracts as an alternative form of treatment and prevention of malarial and various other related medical conditions (6). This could be attributed to the proven therapeutic efficacy, easy accessibility and affordability of herbal medications (7-9) or the growing rate of anti-microbial drug resistance to previously susceptible organisms (10-14). While these alternative forms of treatments have yielded results, their side effects when administered for a prolonged period have not been adequately addressed. As a result, there has been increasing interest in understanding the side effects of the prolonged use of these medications.

Astonia boonei, an abundant evergreen plant is one of the commonest medical plants with almost all its parts being used for the treatment of various diseases including malaria, intestinal helminths, hypertension, chronic diarrhoea and poisoning from snake bites (7, 8). In Nigeria and other west and central African countries where the consumption of this plant species as medicinal herb is very rampant, several pharmacological studies have been conducted to determine the efficacy of extracts from different parts of the plant including its leaves, flowers, stem root and bark (13, 14). These studies have revealed anti-malarial, anti-pyretic, analgesic, anti-inflammatory, diuretic, anti-helmintic and anti-hypertensive properties of the plant extract (13). The stem bark of alstonia boonei is currently listed among the anti-malarial drug in the African pharmacopeia.

A study carried out to evaluate the effect of ethanolic extract from alstonia boonei leaves on levels of serum electrolyte in wistar albino rats at dosages ranging from 100 to 400 mg/kg body weight showed no significant changes (p<0.05) in electrolyte balance at all doses administered (10). However, serum creatinine levels were significantly elevated (p<0.05) when doses higher than 400 mg/kg body weight was administered. Several other studies have also been conducted on various parts of alstonia boonei and its efficacy by administering low concentration doses of the extract to various animal specimen (13, 14). On the other hand, a study on its toxicology that reflects its effects for a prolonged use or high dosage of administration has not been adequately addressed. It is of great medical importance to investigate their effects when administered for a prolonged period because their cheap availability in the African nations makes them affordable and viable options for regular treatment of malaria and related diseases. In view of this, we report detailed study conducted to determine the toxicological effects of the prolonged use of ethanolic extract from alstonia boonei stem bark.

The remainder of this paper is organized into four distinct parts. First, the experimental section describes the animal groupings and plant extract preparations, tests methods and statistical analysis procedure used. Second, the results are presented, and third, the discussions are made by comparing this study with previous works. Lastly, our conclusions are briefly stated.

METHODS

Collection and preparation of plant extract and phytochemical screening

Fresh stem barks were collected from a large alstonia boonei tree at a location in Ile-Ife, Osun State Nigeria. The plant material was authenticated at the Herbanium in the faculty of pharmacy, Obafemi Awolowo University and deposited with the specimen number FPI-1886. Thereafter, the cut stem barks were air dried and then ground into fine powder at the drug research and production unit (DRPU), faculty of pharmacy, Obafemi Awolowo University Ile-Ife. About 500 g of the powder was weighed, diluted into two litres (2 L) of seventy percent (70%) ethanol and allowed to stand at room temperature for two days (48 hours). Afterward, the solution was sieved using a cheese cloth to obtain the supernatant which was further concentrated to dryness using a rotatory evaporator. This produced a brown residue that was termed the ethanolic extract (EE) of alstonia boonei. Using the EE, the phytochemical tests were carried out for the presence of phytochemicals responsible for the anti-microbial potential in the stem bark using standard procedure. Screening for the presence of the following chemicals were carried out: alkaloids, saponins, tannins, flavonoids, terpenoids, cardiac glycosides, phlobatannins and anthraquinone. Detailed description of the phytochemical test protocols has been made elsewhere (14)

Animal preparation, grouping and extract administration for toxicological study

Eighteen (18) albino mice having an average weight of 62-120 g were purchased from the animal house, College of Health Science, Obafemi Awolowo University Ille-ife. The animals were housed in a cage, had free access to rat food and water and were acclimatized for 14 days in the animal house under standard environmental condition. Thereafter, the mice were weighted and randomly grouped into three groups (A, B, C) with each group consisting of six mice. Mice in group A served as the control group and were given only distilled water. Mice in group B received 500 mg/kg body weight of the ethanolic extract, whereas mice in group C received 1000 mg/kg body weight of the extract. Ethanolic extract was administered orally to mice in groups B and C every day and after the first seven days, the first set of mice from groups A, B and C were sacrificed, while the remaining were fed for another seven days (now 2 weeks) and then the second set consisting of 2 mice from each group were sacrificed. The extract administration was then discontinued, and the remaining animals were left alone for another week (now 3 weeks in total) to observe for signs of recovery from previous extract examination.

In sacrificing the animals, all institutional guides for the care and use of laboratory animals were followed. The animals were first anaesthetized with diethyl ether, followed by direct cardiac puncture. The blood samples of the animals were then collected into heparinized sample bottles for chemical evaluation and analysis. Cut portions of the brain, spleen and liver of the mice were obtained and kept in 10% buffered formalin for histopathological evaluation which are not included in the current study.

Biochemical evaluation of blood samples

Blood samples were collected from the euthanized animals and centrifuged at 3000 rpm on 90-2 bench centrifuge for 10 minutes. The supernatant obtained with the randox diagnostic kit was stored in the refrigerator for further analysis. From the supernatant, albumin, aspartate aminotransferase (AST), alanine amino transferase (ALT), creatinine concentration, alkaline phosphate (ALP), bilirubin and urea concentration were estimated. Albumin was estimated using bromocresol green (BCG) modified method of Pinnell and Northan (15), AST and ALT were determined using the colorimetric method of Reitman and Frankel (16). Creatinine concentration was determined using Jaffe’s-alkaline picrate method as described by Kume and co-workers (17). ALP was determined according to the method of Sanni and Van Etten (18). Bilirubin concentration was estimated using Gambino modified method of Jendrassik and Groff (19) and urea concentration was estimated using the colorimetric method of Berthelot as modified by Gordon and co-workers (20).

Statistical analysis

Data obtained from biochemical assay were analysed using one-way analysis of variance (ANOVA). The Turkey HSD multiple comparison test was used to verify differences between treatment groups. The result was expressed as mean± standard error of duplicates.

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