Toxicological and Histopathological Effects of Cheese Wood, Alstonia boonei De Wild Stem Bark Powder used as Cowpea Protectant against Cowpea Bruchid, Callosobruchus maculatus (Fab.) [Coleoptera: Chrysomelidae] on Albino Rats.  

Kayode David Ileke1 , Olusola Olasumbo  Odeyemi2 , Michael  Olufemi  Ashamo2
1. Department of Environmental Biology and Fisheries, Faculty of Science, Adekunle Ajasin University, PMB 001, Akungba-Akoko, Ondo State, Nigeria
2. Department of Biology, School of Science, Federal University of Technology, PMB 704, Akure, Ondo State, Nigeria
Author    Correspondence author
International Journal of Molecular Medical Science, 2014, Vol. 4, No. 2   doi: 10.5376/ijmms.2014.04.0002
Received: 20 Mar., 2014    Accepted: 10 May, 2014    Published: 17 Jun., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Toxicological and histopathological effects of Alstonia boonei stem bark powder on albino rats liver and kidney functions were investigated using standard methods. After two weeks of acclimatization, the rats were randomly divided into four (I - IV) groups of six animals each. Group I was fed with Basal diet, group II - IV were for 30 days with basal diet containing 1%, 4% and 10% A. boonei stem bark powder, respectively. Blood was rapidly collected by direct heart puncture and the plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, urea and creatinine contents were determined using commercially-available kits. The levels of these biochemical functions were also determined in the liver and kidney. The A. boonei stem bark powder at 10% concentration caused oxidative liver and kidney damages. Histopathological results of the liver shows that the group fed with 4% A. boonei stem bark powder had normal hepatocytes. The group fed with 10% A. boonei stem bark powder had necrosis. The results of the present study showed that the supplementation of 1% and 4% A. boonei stem bark powder to the diets of rats for 30 days did not change the biochemical parameters of liver and kidney as well as histopathological investigation which illustrated normal architecture of liver and kidney.
Keywords
Alstonia boonei; Albino rats; Callosobruchus maculatus; Cowpea seed; Toxicology; Histopathology

 1. Introduction

Alstonia boonei De Wild (Apocyanaceae) is an African large evergreen deciduous crude medicinal tree that shed its leaves annually. The plant is about 45 m tall and 1.2 m in diameter. It possess roots, stems, barks, leaves, fruits, seeds, flowers, and latex, which are claimed to have medicinal values in some cultures in African countries. The plant stem bark and its latex are applied in traditional medicine for treating many diseases. There are records on the use of alcoholic or aqueous extracts of most parts of A. boonei (Moronkola and Kunle, 2012). In traditional African medicine, A. boonei is a medicinal plant used extensively for the treatment of malaria, fever, intestinal helminths, rheumatism, hypertension (Terashima, 2003; Bett, 2004; Abel and Busia, 2005). Itis used for the treatment of chronic diarrhea and dysentery, fever, pain, intestinal disorders and as an antidote for Strophanthus poison (Amole and Ilori, 2010). The extracts of the stem bark are commonly used to treat malaria and is listed in the African Pharmacopoeia as an antimalaria drug (Olajide et al., 2000). An infusion of root and stem bark is drunk as a remedy for asthma. A liquid made from the stem bark and fruit is drunk once daily to treat impotence (Majekodunmi et al., 2008). The stem bark is used for the treatment of febrile illness, painful urination, rheumatic conditions and jaundice (Asuzu and Anaga, 1991), malaria, fever (Bello et al., 2009; Majeko- dunmi and Odeku, 2009; Majekodunmi et al., 2008), intestinal helminths (Weshche et al., 1990), rheumatism, reversible antifertility (Raji et al., 2005) and hypertension (Olajide et al., 2000; Abel and Busia, 2005; Betti, 2004; Terashima, 2003) as an anti-venom against snake bite and antidote against arrow poisoning (Moronkola and Kunle, 2012). Other pharmacological uses are anti-inflammatory and as analgesic (Olajide et al., 2000).

The major phytochemicals in the stem bark are saponins, alkaloids, tannins, flavonoids and cardiac glycosides (Anon, 1992; 2001; Phillipson et al., 1987).
In addition, it has also been suggested that it also contains macro elements such as calcium, magnesium, sodium, potassium, phosphorus, iron, zinc, manganese, copper and cobalt to varying degrees (Akinmoladun et al., 2007; Amole and Ilori, 2010). Alkaloids are medicinally useful, possessing analgesic, antispasmodic and bactericidal effects. Tannins promote healing (Oliver, 1960; Okwu and Okwu, 2004). Cardiac steroids are widely used in treating congestive heart failure (Okwu and Okwu, 2004). Flavonoids lower risk of heart diseases, saponins also promote wound healing (Okwu and Okwu, 2004).

The insecticidal activity of A. boonei has been reported recently (Oigiangbe et al., 2007a; 2007b; Ileke and Oni 2011; Ileke et al., 2012; Omoya et al., 2012;  Ileke et al., 2013; 2014a; 2014b; 2014c; Ojo and Ogunleye, 2013). Aqueous extracts of the leaf and stem bark of A. booneiwas active against the pink borer, Sesamia calamistis Hampson (Lepidoptera: Noctuidae), a pest of maize and some other cereals in West and Central Africa (Oigiangbe et al., 2007a). Ileke and Oni (2011) reported the insecticidal potential of plants including A. boonei against Sitophilus zeamais. Ileke et al. (2012 and 2013) reported the insecticidal activity of A. boonei powder against C. maculatus and response of cowpea bruchid to A. boonei stem bark oils extracted with methanol, ethanol, acetone, petroleum ether and n-hexane. Omoya et al. (2012) reported the insecticidal potential of A. boonei leaf extract against Anopheles mosquito larvae in Nigeria. Ojo and Ogunleye (2013) worked on the comparative effectiveness of the powders of some underutilized botanicals including A. boonei stem bark powder for the control of Sitophilus zeamais. Ileke et al. (2014a) reported the insecticidal activity of A. boonei latex against C. maculatus. We present the toxicological and histopathological effects of A. boonei stem bark powder which is scarce in literature.

2. Materials and Methods
2.1 Preparation of Alstonia boonei  
Stem bark of Alstonia boonei used for this study was sourced fresh from Akola farm at Igbara-Odo Ekiti, Ekiti State, Nigeria. The collected stem bark was rinsed in clean water to remove sand and other impurities, cut into smaller pieces before air-dried in the laboratory. The cleaned dried plant parts were pulverised into very fine powder using an electric blender (Supermaster ®, Model SMB 2977, Japan). The powders were further sieved to pass through 1mm2 perforations (Ileke and Oni, 2011). The powder was packed in plastic containers with tight lids and stored in a refrigerator at 4℃ prior to use.

2.2 Toxicological investigation of A.  boonei stem bark powder
2.2.1 Animals
Adult female albino rats weighing 160–170g were purchased from the breeding colony of the Department of Biochemistry, Federal University of Technology, Akure, Nigeria. The rats were maintained at 25℃ on a 12 hours light/dark cycle with free access to food and water. They were acclimatized under these conditions for two weeks prior to the commencement of the experiments.

2.2.2 Feed formulation and treatment groups
The diets were freshly formulated according to the modified method of Oboh (2005) and were kept in air tight containers and stored at 4℃ until needed for use. Animals were divided into four groups:
·         Group I – control rats, fed with basal diet (44% skimmed milk, 42% corn starch, 4% mineral and vitamin premix and 10% groundnut oil);
·         Group II – rats fed with basal diet plus 1% A. boonei stem bark powder for 30 days;
·         Group III – rats fed diet supplemented with 4% A. boonei stem bark powder for 30 days; and
·         Group IV – rats fed diet supplemented with 10% A. boonei stem bark powder for 30 days.
2.2.3 Alstonia boonei stem bark powder fed bioassay
Control and treated animals were decapitated after an overnight fast by cervical dislocation. The blood was rapidly collected by direct heart puncture and plasma was prepared using standard method. Also, the plasma AST, ALT, ALP, total protein, urea and creatinine contents were determined using commercially- available kits (Randox Laboratories, UK).
2.3 Preparation of tissue homogenates
Liver and kidney of the rats were rapidly isolated and placed on ice and weighed. Tissues were rinsed in cold 0.9% normal saline (1:3, w/v), subsequently homogenized in sodium phosphate buffer (pH 6.9) and the homogenates centrifuged. The clear supernatants obtained were used for various biochemical assays (Belle et al., 2004).
2.4 Histological analysis of liver and kidney of rats fed with A. boonei stem bark powder 
The sectioning method described by Akparie (2004) was used for the histological examination. Sections of the liver and kidney 6µm thin were made and studied under the microscope.  This method has the advantage of preserving the relations of cells and tissues to one another. They were dehydrated in serial concentration (50, 70, 80, and 100%) of alcohol 1½ hour each. After dehydration, they were cleared with 100% xylene and were left for 2 hours to remove any remnant alcohol, and later impregnated in liquid wax for 2 h for embedding. The embedded organs were sectioned using microtome and were stained with haematoxylin-eosin (Silva et al., 1999). Excess stain was removed with tap water. After clearing in xylene, Canada balsam was added and cover slips placed on the slides. The preparations were left in the oven at 40°C and then placed under the microscope equipped with a digital camera connected to a computer system to be examined by a Histopathologist and the photographs were taken.
2.5 Data Analysis
Data were subjected to Analysis of Variance (ANOVA) and treatment means were separated using the New Duncan’s Multiple Range Test. The ANOVA was performed with SPSS 16.0 software (SPSS, Inc. 2007).
3. Results
3.1 Feeding intake of all experimental animals
The average feed intake (Table 1) of all the experimental animals were calculated after the 30 days of study and it was discovered that, there exist no significant (P>0.05) difference in the average feed intakes of normal control rats (normal rats fed basal diets), rat fed with basal diet plus 1%, 4% and 10% A. boonei stem bark powder.
 
Table 1 Average Feed Intakes of Rats Fed Diets with Basal Diet and Basal Diet plus A. boonei Stem Bark Powder
Group
Average feed intake (g/rat/day)
I
9.23+1.37a
II
9.07+1.50a
III
8.43+1.92a
IV
8.01+1.73a
Note: Each value is a mean + standard error of six replicates. Means followed by the same letter along the column are not significantly different (P>0.05) using New Duncan Multiple Range Test.
3.2 Effect of A. boonei stem bark powder on body weight of Rats
Mean values for rats’ body weight fed with basal diet and basal diet plus A. boonei stem bark powder at various doses for 30 days period is presented in Table 2. Measurement of the body weight was used to evaluate the health status of the rats during the experimental period. There was no significant difference (p<0.05) in the body weights of rats from the start until the end of the experimental period in all groups apart from rat fed with basal diet plus 10% A. boonei stem bark powder that show weight loss compared with the normal control rats (basal diet only), rat fed with basal diet plus 1% and 4% A. boonei stem bark powder.
 
Table 2 Change in Body Weight of Rats Fed with Basal Diet and Basal Diet plus A. boonei Stem Bark Powder
Groups
Initial weight (g)
Final weight (g)
Weight gain/loss (%)
I
164.23±9.21a
176.67±10.37b
7.61b
II
165.67±9.52a
175.23±9.21b
5.73b
III
167.53±9.43a
171.67±9.52b
2.51b
IV
163.33±9.19a
157.13±9.79a
-3.80a
Note: Each value is a mean + standard error of six replicates. Means followed by the same letter along the column are not significantly different (P>0.05) using New Duncan Multiple Range Test.
 
3.3 Effects of A. boonei stem bark powder on liver functions of albino rats
Tables 3 and 4 presented the effects of A. boonei stem bark powder on liver and serum biochemical indices of Albino rats respectively. The liver activities of aspartate aminotransferase (AST), alanine amino- transferase (ALT), alkaline phosphatase (ALP) and total proteins (TP) of the animals fed with basal diet (Group I), animals fed with 1% and 4% A. boonei stem bark powders were significantly higher than the animal fed with 10% A. boonei stem bark powder (Table 3). However, there were significant increases in the activities of these enzymes in the serum of the animal fed with 10% A. boonei stem bark powder as compared with the control, animals fed with 1% and 4% A. boonei stem bark powders (Table 4). Increase in the serum enzyme activity signifies damages to the liver membrane. The serum AST activity of animal fed with 1% and 4% A. boonei stem bark powders were not significantly different form each other, whereas the serum AST activity of the group fed with 10% A. boonei stem bark powder only (78.67 IU/g) was significantly higher than that of the control (35.43 IU/g) (Table 4). Generally, the activities of these enzymes in the animal fed with 1% and 4% A. boonei stem bark powders groups compared favourably with the animals fed with basal diet (Tables 3 and 4).


Table 3 Effects of A. boonei Stem Bark Powder on some Liver Biochemical Indices of Rats.
Groups
AST IU/g
ALT IU/g
ALP IU/g
Total protein min (x 10-1)
I
194.33±2..59b
156.71±3.19b
52.33±2.65b
1.83±0.01a
II
189.67±2.37b
153.33±3.24b
48.67±2.61b
International Journal of Molecular Medical Science
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