Comparative Antibacterial Efficacy of Vitellaria paradoxa ( Shea Butter Tree ) Extracts Against Some Clinical Bacterial Isolates

The antibacterial activities of the ethanolic extracts of seed, leaf and stem bark of Vitellaria paradoxa were investigated. The extracts were tested against three clinical bacterial pathogens, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae using the agar diffusion and the broth dilution techniques. Ethanolic extracts of the plant parts showed activity against all the bacterial pathogens tested. At the highest extract concentration (200 mg/ml), the leaf extract exhibited the highest antimicrobial activity, while no activity was detected at the lowest concentration (3.13 mg/ml) against the tested isolates. Escherichia coli and Staphylococcus aureus were more susceptible to all extracts of V. paradoxa, while Klebsiella pneumoniae showed the least sensitivity. The efficacy of ethanolic extracts of Vitellaria paradoxa was compared to a commercial antibiotic streptomycin. There were differences in the minimum inhibitory concentration (MIC) of all the Vitellaria paradoxa ethanolic extracts with respect to the type of organism. All extracts exhibited bacteriostatic effects against the tested organisms at the experimented concentrations. Qualitative phytochemical screening of the extracts revealed the presence of saponins, tannins and alkaloids as the active principles of Vitellaria paradoxa's antimicrobial activity. V. paradoxa could be used as a potential source of antibiotic substance for a drug development.


Introduction
Traditional medicines have been effectively used for thousands of years.The contribution of herbal products to modern medicine is well documented.It is reported that life in most parts of Africa is connected with herbal medicine, while 65%-80% of world's population rely on traditional medicine for their health care needs (Philips et al., 2009;Calixto, 2000;Adamu et al., 2013).In Nigeria, thousands of plant species are known to have medicinal values and the use of different parts of these plants to cure specific ailments has been practiced since ancient times (Rios and Recios, 2005).The medicinal values of plants lie in their phytochemical composition, which produce definite physiological actions on the human body (Mann et al., 1997).
V. paradoxa (formerly Butryspermum paradoxum) or Shea butter, is a popular tree with several applications in folkloric medicine.The tree grows naturally in the wild of the dry savannah belt of West Africa and stretches in abundance onto the foothills of the Ethiopian mountains (Adamu et al., 2013).
In traditional medicine, Shea butter has been employed in the treatment of several ailments.It encourages wound healing and soothes skin irritation.Shea-butter is also used to treat inflammation, rashes in children, dermatitis, chapping and ulcers, as well as rub for rheumatism (Hong et al., 1996).Its leaf decoctions are used for stomach ache, headache and as an eye lotion.Roots and root bark are grounded to paste and taken orally to cure jaundice, or they are boiled and pounded to treat chronic sores.They are also used for the treatment of gastric problems as well as diarrhea and dysentery.Bark decoction is used to facilitate childbirth and to encourage lactation after delivery, or as a footbath neutralizes venom of the spitting cobra (Hall et al., 1996).Cosmetics, especially those that prevent skin drying and good looking lipsticks use Shea-butter.As a result, cosmetic industries market uses these ingredients in soaps, shampoo and skin cream preparations (Hall et al., 1996).
V. paradoxa has been studied as a potent medicinal plant (Prescott et al., 2002), against bacterial infections (El-Mahmoud et al., 2008) and fungal infections (Ahmed and Sani, 2013).The ethanolic extraction of the active principle of this medicinal plant has been shown to be more efficacious than when water or acetone (El-Mahmoud et al., 2008) and hot or cold water (Ahmed and Sani, 2013) are used as extractants.The reason for the higher antibacterial activity of the ethanolic extract has been suggested to be due to differences in the polarity of the solvents and the modulatory effect of enzyme such as phenolases and hydrolases released when plant materials are grounded in water (El-Mahmoud et al., 2008).Odebiyi and Sofowora (1978) also showed that potency of a plant extract depends on both the concentration used and the method of extraction.
In line with the need to search for more effective and safe antibacterial drugs and to justify the traditional use of herbal preparations in the treatment of infectious diseases, this work was designed to investigate and compare the antibacterial efficacy of the Vitellaria paradoxa leaf, seed and stem bark against some clinically important isolates and to determine the phytochemical constituents present in those plant extracts.

Test organisms
The bacterial species used in this study, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, were obtained from the Department of Microbiology, University of Ilorin Teaching Hospital (UITH), Ilorin, Kwara state.Purity of the cultures was checked at regular intervals as described by Acheampong et al. (1988).

Plant collection and identification
Samples of the bark, seed and leaf of Vitellaria paradoxa were collected from the trees within University of Ilorin, Ilorin, Nigeria.The plant samples were identified macroscopically as described by Dalziel (1968) and confirmed at the herbarium unit of the Department of Plant Biology, University of Ilorin.The fresh samples were sundried for a week, grounded into a fine powder and kept in plastic containers until further use at room temperature (28 ± 1 °C).

Preparation of ethanolic extracts
One hundred grams (100 g) of each of the plant parts (seed, leaf and stem bark) were soaked into 100 ml of the solvent (95% ethanol) in different air-tight sterile jars respectively at room temperature and kept on a shaker (90 rpm) with uniform shaking for 24 hours.The solvents containing the extracts were decanted filtered with a muslin cloth and then with Whatman no. 1 filter paper respectively.Further extraction of the grounded samples was done with same volume of 95% ethanol, decanted and filtered two more times.The filtrates from each round of extraction were combined and were evaporated to dryness in small, openmouth jars and then packed in separate clean dry bottles and stored at room temperature until required.

Sterility of extracts
Each of the extracts was tested for growth of contaminants.This was done by making serial dilution of 1 g of each extract up to 10 -1 .Twenty microliters (20 µl) of the diluents were aseptically inoculated on Nutrient Agar plates and incubated at 37 °C for 24 hours.The plates were observed for growth.Absence of microbial growth in the extract indicated their sterility.Sterile extracts were used to test for antimicrobial efficacy.

Standardization of inoculums
Standardized inoculums of each tested organism was obtained by making their respective suspension up to 0.5 McFarland standard as observed in the spectrophotometer and as described by Barry et al. (1980).

Determination of antimicrobial activities
The Agar Well Diffusion method as described by Lino and Deogracious (2006) was used.By this method, 0.1 ml of the respective standardized inoculums (0.5 McFarland turbidity standard = 1.0 x 10 8 cfu/ml) of each test bacterium was spread into sterile Mueller Hinton Agar plates so as to achieve even growth.The plates were allowed to dry and a sterile cork borer (5.0 mm diameter) was used to bore wells aseptically in the agar plates.The extracts were prepared and serially diluted in a two-fold dilution to achieve different concentrations of 3.13, 6.25, 12.5, 25, 50, 100 and 200 mg/ml respectively for each extract.Subsequently, 0.3 ml of each concentration of the extracts was introduced into the wells earlier bored Agar plates.The extracts were allowed to diffuse into the medium (kept for 1 hour on the bench before incubation) at 37 °C for 24 hours.Streptomycin was used as a positive control, while a Mueller Hinton agar plate without antimicrobials was the negative control.Antimicrobial activity of the extracts was determined by measurement of zones of inhibition produced around the wells.The diameter of the zones indicated the degree of susceptibility of the test bacteria.

Determination of Minimum Inhibitory Concentration (MIC)
The MIC of the ethanolic extracts against the test organisms was determined using the broth dilution method described by Sahm and Washington (1990).Briefly, 1.0 ml of the extract solutions (seed, leaf and bark) at concentrations of 200 mg/ml, 100, 50, 25, 12.5, 6.25 mg/ml and 3.13 mg/ml were added to 9 ml of sterile nutrient broth in different test tubes respectively.100 µl of an 18 hours culture adjusted to 0.5 McFarland turbidity standard (1.0 x 10 8 cfu/ml) was inoculated in each test tube.The tubes were incubated at 37 °C for 24 hours.Four control tubes were set up for each test batch of organism.These included the antibiotic control (three tubes containing the respective extracts and the growth medium without the inoculums) and a blank (a tube containing only sterile nutrient broth).The tube with the lowest concentration of the extracts (highest dilution) which had no detectable bacterial growth when compared with the control tube (using both physical and spectrophotometer observations) was considered the Minimum Inhibitory Concentration (MIC).

Determination of Minimum Bactericidal Concentration (MBC)
The Minimum Bactericidal Concentration of the extracts was determined by subculturing test solutions which showed no detectable growth (no turbidity after 24 hours incubation) onto fresh Nutrient Agar plates (the recovery medium) and incubated further for 24 hours.Absence of growth on the recovery medium indicated bactericidal effect, while the appearance of growth on further incubation indicated bacteriostatic effect.

Phytochemical screening of ethanolic extract
The extracts were screened for the presence of carbohydrates, tannins, alkaloids, saponins, polyphenols and other constituents successively as described by Odebiyi and Sofowora (1978) and Herbune (1973).

Results and Discussion
The current experiment investigated and compared the antimicrobial activity of the ethanolic extracts of leaf, bark and seed of V. paradoxa (Shea butter tree) against selected clinical bacterial isolates (Staphilococcus aureus, Klebsiella pneumoniae and Escherichia coli.V. paradoxa) as it has recently been a research focus as potential source for drug development due to its antibacterial (El-Mahmood et al., 2008) and anti fungal (Ahmed and Sani, 2013) activities.

Phytochemical composition
Phytochemical composition of V. paradoxa is shown in Table 1.General glycosides, tannins, saponins, carbohydrates, alkaloids and trepenoids were present in all plant parts (stem bark, root and leaf) of V. paradoxa ethanolic extracts.Steroids were absent in both the stem bark and seed, while leaf extract was shown to lack polyphenols.These bioactive compounds have been demonstrated to be responsible for the antimicrobial activity of medicinal plants (Mathias et al., 2007).

Effect of ethanolic extracts of V. paradoxa parts against selected organisms
As shown in Tables 2, 3 and 4, all the plant parts tested, the bark, leaf and root respectively, showed antibacterial activity against the selected clinical isolates.The leaf extract showed the highest activity against all tested organisms and its efficacy was comparable to the commercial antibiotic (streptomycin) at the highest concentration (200 mg/ml) against test organisms.This higher activity is thought to be premised upon the presence of steroids, which was absent in both the bark and the seed extract.The absence of steroids in the bark of V. paradoxa has been reported (El-Mahmood et al., 2008)   13.5 ± 00 12.5 ± 03 9.0 ± 02 18.0 ± 03 100 9.5 ± 03 6.0 ± 02 7.0 ± 00 16.0 ± 02 50 7.0 ± 01 5.0 ± 03 3.5 ± 01 15.0 ± 03 25 7.0 ± 01 3.5 ± 03 2.0 ± 02 14.0 ± 01 12.5 4.5 ± 03 0.5 ± 02 1.5 ± 02 12.0 ± 00 6.25 3.0 ± 01 --6.0 ± 01 3.13 ---3.0 ± 01 Klebsiella pneumoniae McFarland turbidity standard (1.0 x 10 8 cfu/ml) response to the respective extracts at different concentrations.Leaf and bark extracts were more potent against E. coli and S. aureus, while Klebsiella pneumoniae was more susceptible to bark extract treatment.Among the three isolates, E. coli was the most susceptible organism to all V. paradoxa extracts (Tables 2, 3, 4).Klebsiella pneumoniae was the least susceptible, while Staphylococcus aureus was susceptible, but slightly less susceptible as E. coli.The differences in the susceptibility of the tested organisms and variations in the specific activity of each extract may be due to the physiological properties of the clinical isolates and the presence or absence of some active principles in the extracts.More so, the sensitivity of the organisms, as indicated by the diameter of the inhibition zones (Tables 2, 3, 4) was proportional to the concentration of the respective extracts.At the highest concentration (200 mg/ml), all organisms were sensitive to the V. paradoxa extracts.Progressive decrease in respective extract concentration leaded to a proportionate reduction of the inhibition zone around each organism.Similar reports (Adamu et al., 2013;Arekemase et al., 2013) have shown that higher concentrations of antimicrobial substances showed appreciable antimicrobial activity.
All V. paradoxa extracts exhibited bacteriostatic effects (Table 5) on all tested clinical isolates, but showed different minimum inhibitory concentration (MIC) which was also different with respect to each organism tested in the experiment.The bacteriostatic effect of these plant parts could possibly be due to the presence of saponins, which demonstrate remarkable physiological activity and forms lather, responsible for wound and skin protection (Ahmadu et al., 2006).Specifically, saponins have been suggested to exhibit greater antimicrobial effect and could serve as a precursor of steroidal substances with a wide range of physiological activities.The MIC of the stem bark extract was 6.25 mg/ml against all the organisms, while the seed and leaf extracts had MIC of 6.25 mg/ml against E. coli and S. aureus, but 12.5 mg/ml against K. pneumonia respectively.However, at 12.5 mg/ml, all V. paradoxa extracts showed antibacterial activity against all the clinical isolates.The variation in the MIC may be due to the phytochemical composition of the respective ethanolic extracts and the genetic make-up of each test organisms.Different organisms have been shown to respond differently to different and same concentrations of a specific medicinal plant (Philip et al., 2009).
As shown in Table 5, the plant extracts were not bactericidal on the tested organisms at all the concentrations.This was demonstrated by the re-growth of the organisms when samples taken from around the clearance zone were cultured on fresh nutrient agar plates.

Conclusions
The ethanolic extracts of the bark, seed and leaf of Vitellaria paradoxa have demonstrated antimicrobial activities against the tested clinical isolates (E.coli, S. aureus and Klebsiella pneumonia), thus justifying its use in traditional medicine for treating different diseases associated with the tested isolates, as it also could serve as a new and cheaper alternative for antibiotic sources.The clinical isolates used for this investigation are associated with various human diseases like gastrointestinal tract infections, pneumonia and body superficial wound infections.As shown in this study, V. paradoxa could be used as a potential source of antibiotic substance for a drug development against the diseases caused by this group of both superficial and enteric organisms.Further toxicological, purification and identification studies could be carried out to investigate the general effects of the use V. paradoxa for drug development.
Concentration (mg/ml) Diameter of inhibition zones of V. paradoxa extracts and commercial antibiotic (mmStaphylococcus aureus McFarland turbidity standard (1.0 x 10 8 cfu/ml)Table 3. Antimicrobial activity of V. paradoxa extracts against Escherichia coli Concentration (mg/ml) Diameter of inhibition zones of V. paradoxa extracts and commercial antibiotic (mm) Escherichia coli McFarland turbidity standard (1.0 x 10 8 cfu/ml)

Table 1 .
Phytochemical screening of crude extracts of Vitellaria paradoxa

Table 2 .
Antimicrobial activity of V. paradoxa extracts against Staphylococcus aureus

Table 4 .
Antimicrobial activity of V. paradoxa extracts against Klebsiella pneumoniae

Table 5 .
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of S. aureus, E. coli and K. pneumonia