MATERIALS AND METHODS

Collection of plant materials: Samples of the stem bark of Vitellaria paradoxa were collected in April from the Permanent Site Campus of the University of Ilorin, Nigeria. The stem bark samples were authenticated at the Herbarium Unit of the Department of Plant Biology, University of Ilorin, Nigeria where voucher specimen was deposited. The study was conducted between April, 2011 and July, 2011.

Animals: Healthy Swiss mice (20-25 g) and albino Wistar rats (180-200 g) used in the study were obtained from the Animal House of the College of Health Sciences, Ladoke Akintola University of Technology, Ogbomoso, Nigeria. They were maintained under standard laboratory conditions of humidity (40-60%), temperature (21±1°C) and light (12/12 h light/dark cycle). The animals were acclimatized for one week, fed on rat pellets (Livestock Feeds PLC, Ibadan, Oyo State, Nigeria) and allowed free access to drinking water ad libitum. They were fasted overnight before the experiment was carried out. All conditions of animal use were as approved by United States National Institute of Health (NIH) guide for Care and Use of Laboratory Animals and in accordance with the recommendation of IASP (Zimmermann, 1983).

Preparation of the plant extracts: The stem bark of V. paradoxa was air-dried under shade to constant weight for 14 days and the dried materials were chopped into small pieces using a pestle and mortar and then pulverized into powdery form using an electric blender. One hundred grams of the powdered plant material was soaked in 500 mL of distilled water or 95% ethanol in separate 1.5 L sterile conical flasks at room temperature. These were subjected to uniform agitation in a shaker water bath for 48 h. The content was then filtered with a piece of muslin cloth and then Whatman filter paper (No. 1). The filtrates were evaporated to dryness and the resultant extracts were packed in separate clean dry bottles and stored in a refrigerator at 4°C until use. The yields of aqueous and ethanol extracts are 0.6 and 0.8%, respectively.

Collection and identification of bacteria: The clinical isolates of E. coli, K. pneumonia, P. mirabilis, E. faecalis, S. aureus, P. aeruginosa and S. pyogenes were collected in peptone water from the Microbiology Laboratory of Nigeria Institute of Medical Research, Lagos, Nigeria Preliminary identification of the test organisms were carried out in the Microbiology Laboratory of LAUTECH Teaching Hospital, Osogbo, Nigeria. The organisms were stored on Nutrient agar slants in a refrigerator (2-8°C). Purity of the cultures was checked at regular intervals as described by Acheampong et al. (1988).

Determination of antimicrobial activity: Antimicrobial activities of aqueous and ethanolic extracts of Vitellaria paradoxa were carried out using the agar well diffusion method (Vanden Berghe and Vlietnck, 1991). The bacterial strains grown on nutrient agar at 37°C for 18 h were suspended in a saline solution (0.9%, w/v) NaCl and adjusted to a turbidity of 0.5 McFarland standard (10⁸ CFU mL^-1). To obtain the inocula, these suspensions were diluted 100 times in Muller Hinton broth to give 10⁶ colony forming units (CFU) mL^-1. Standardized inoculum of each test bacterium was spread onto nutrient agar plates so as to achieve even growth and the plates were allowed to dry. Discs were obtained using Whatman filter paper (No. 1). They were prepared by punching and then put in vials-bottles and sterilized in an oven at 150°C for 15 min. Thereafter the discs (9 mm diameter) were aseptically bored into the solid nutrient agar using a sterile cork borer. The test solutions of extracts at concentration of 50 mg mL^-1 were then introduced into each of the designated discs on each plate while ensuring that no spillage occurred. Sparfloxacin at a concentration of 5 μg mL^-1 (Solidum Pharmaceuticals, Nigeria) was used as a positive control and distilled water as negative control. The plates were left at room temperature for 1 h, allowed to diffuse into the medium, turned upside-down and thereafter incubated at 37°C for 24 h in an incubator. Clear zones of inhibition were observed. Activity of each extract was tested in triplicate and the diameters of zones of inhibition were measured in millimeter.

Acute toxicity test: The ethanol extract of V. paradoxa was tested because of its higher antibacterial activity against all the bacteria considered. Mice were divided into six groups of six animals each. The extract was administered to mice in groups 2-6 in single oral doses of 20, 40, 80, 160, 320 and 640 mg kg^-1 b. wt., respectively by intra gastric gavage using oral cannula (a feeding needle). The control group (group 1) received an equal volume of distilled water as vehicle. Observations of toxic symptoms were made and recorded within 24 h after administration of the extract. Behavioral parameters and mortality were monitored closely for the initial 2 h and thereafter for 24 h. Lethal dose in fifty per-cent of the total population (LD₅₀) was interpolated using Lorke (1983) method.

Effects on hematological and biochemical parameters: Ten albino Wistar rats were used for the study. They were randomly divided into 2 groups of 5 rats each. Group 1 served as the control and received normal saline (5 mL kg^-1). Group 2 was treated with 12 mg kg^-1 of the extract for 28 days. All drugs were administered orally. On the 29th day, rats were anaesthetized in a glass chamber containing cotton wool soaked in diethyl ether and blood samples were collected by cervical decapitation into clean lithium heparin bottles. The blood samples were centrifuged for 5 min and the supernatant plasma was subsequently used for the assay of biochemical parameters while whole blood was used for hematological assays. An automated hematologic analyzer (Sysmex KX-21, Japan) was used to analyze the hematological parameters of Hemoglobin (Hb), Packed Cell Volume (PCV), Red Blood Cell (RBC), Hematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC), White Blood Cell (WBC), neutrophils, lymphocytes and platelets. AST, AL, ALP, serum urea and creatinine levels were determined as described by Toro and Ackermann (1975).

Statistical analysis: All data were expressed as Mean±SEM and statistically analyzed by Student’s t-test at 95% confidence limit.

RESULTS

Oral dosing of 20, 40, 80, 160, 320 and 640 mg kg^-1 of the ethanol extract resulted in different percentage death of the rats as shown below (Table 4). LD₅₀ was calculated as 115 mg kg^-1 from a curve of log dose vs. %response. Behavioral changes observed include reduced motor activity, increased breathing rate and abdominal writhes.

Both aqueous and ethanol extracts demonstrated antimicrobial activity against five out of the seven bacteria strains but ethanol extract demonstrated higher antibacterial activity especially on K. pneumoniae, E. faecalis and S. aureus. P. aeruginosa and S. pyogenes were completely resistant to both extracts. These results are presented in Table 1.

Following the treatment of rats with ethanol extract of V. paradoxa for 28 days, ALP, ALT, AST, creatinine and urea plasma levels of the treated rats increased significantly (p<0.05) compared with the control (Table 2). There was no significant difference between the control and test groups in the haematological indices tested except the platelets which significantly decreased in the test group (Table 3).

Table 1:	Antibacterial activity of stem bark extract of Vitellaria paradoxa
Value are represents Mean±SEM for three trials, na: Non-measurable zone of inhibition, ap<0.05 compared with negative control which has no antibacterial activity

Table 2:	Effects of stem bark ethanol extract of Vitellaria paradoxa on biochemical parameters in rats
Values are Mean±SEM (n = 5), ap<0.05 compared with control

Table 3:	The effects of stem bark ethanol extract of Vitellaria paradoxa on haematological parameters in rats
Values are Mean±SEM (n = 5), *p<0.05 compared with the control

Table 4:	Acute toxicity of ethanol extract of Vitellaria paradoxa stem bark

DISCUSSION

Ethanol extract of V. paradoxa exhibited inhibitory effect on the growth of E. coli, K. pneumonia, P. mirabilis, E. faecalis and S. aureus but not on P. aeruginosa and S. pyogenes. The organisms that are susceptible to the extract are known to cause many life-threatening diseases such as meningitis, endocarditis urinary tract infections and gastrointestinal tract infections among others (Adebayo and Ishola, 2009). So the extract could be a good alternative in the treatment of these infectious diseases. S. aureus exhibited the highest sensitivity followed by E. faecalis. This is an indication that Gram-positive bacteria are more susceptible to the antimicrobial effect of V. paradoxa than Gram-negative bacteria. This is consistent with previous studies on other medicinal plants (Ceylan and Fung, 2004; Lopez et al., 2005). The significant differences in the outer layers of Gram-negative and Gram-positive bacteria may be responsible for this observation. The resistance of Gram-negative bacteria towards antimicrobial agents is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharide molecules, presenting a barrier to the penetration of numerous antibiotic molecules (Shan et al., 2007). It is also associated with the enzymes in the periplasmic space, which are capable of breaking down the molecules introduced from outside (Russel, 1991). Gram-positive bacteria lack this outer coating and cell wall. Therefore the cell wall of Gram-positive organisms is more prone to destruction by antibacterial agents. However it should be noted that the extract also exerted some moderate activity on Gram-negative bacteria used for the study except in P. aeruginosa in which it was completely devoid of activity. The sensitivity of K. pneumoniae, P. mirabilis and E. coli to the extract agrees with the work of El-Mahmood et al. (2008). Ethanol extract of the plant has higher antibacterial effect than water extract. This is probably because ethanol extracted more of the phytochemical constituents of the plant that are responsible for its antibacterial activities.

Phytochemical studies of stem bark of V. paradoxa revealed the presence of alkaloids, tannins, saponins, steroids and carbohydrates (Ndukwe et al., 2007). These bioactive compounds are likely responsible for the antimicrobial activity of the plant against the organisms (Ndukwe et al., 2005). There was significant increase in the plasma levels of ALT, AST and ALP of rats treated with ethanol extract of the plant for 28 days. Increase in the levels of these enzymes is an indicator of tissue damage and altered membrane permeability (Kolawole et al., 2011). Damage to liver, kidney, bone and small intestine may increase the level of ALP in blood (Adedapo et al., 2007). Increase in creatinine and urea levels is an indication of injury to the kidney (Aliyu et al., 2007).

The platelets count in the treated rats was significantly lower (p<0.05) than that of the control. Platelets are involved in the regulation of clotting when there is injury to the blood vessel walls. When platelet count is low, clot retraction is deficient and there is poor contraction of ruptured vessels (Ganong, 2002) which results in increasing bleeding tendency after a minor injury.

CONCLUSION

This work has demonstrated that Vitellaria paradoxa stem bark extract possesses broad spectrum antibacterial activity against some clinical isolate of pathogenic bacteria. The finding justifies the use of the plant in traditional treatment of wounds, leprosy, oral infection and other infectious diseases and therefore has promising potential to provide lead molecules for the development of novel antibacterial drugs. The study also showed that prolonged administration of the plant extract at high doses may adversely affect the functions of some vital organs. Further study of the plant is necessary to isolate the active principles and determine the mechanism of action.