Phytochemical screening and in vitro evaluation of antibacterial activity of aqueous and ethanolic extracts of root and stem bark of Bridelia ferruginea. Benth. (Euphorbiaceae)

This study aimed at analysing the phytochemical content and antimicrobial activity of Bridelia ferrugine against selected bacteria. Total saponin, alkaloids, tannins, flavonoids and total anthraquinone contents were evaluated using spectrophotometric equivalents of the standards. The antibacterial activity of the plant extracts were determined using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays against selected bacteria. The root and stem revealed the presence of the phytochemicals tested except the stem that lacked anthraquinones. In vitro antimicrobial activity of the plant extracts against the gram-positive bacteria tested showed that Bacillus cereus was most susceptible to the plant extract having MIC and MBC of 25 and 50 mg/ml, respectively for the stem-bark and root-bark ethanolic extract, while gram-negative bacteria the plant extracts were most active against Proteus mirabilis with MIC and MBC of 50 and 100 mg/ml, respectively. The aqueous extract was most active against Staphylococcus epidermidis with MIC and MBC of 50 and 100 mg/ml for stem-bark and 25 and 50 mg/ml for root-bark extract. Concentration dependent study showed the plant extracts were either bacteriostatic or bactericidal. Only the stembark aqueous extract showed no primary effect on the control strains. The study confirmed the presence of some phytochemicals which revealed that the plant is of pharmacological importance going by the ability of these phytochemicals to elicit antibacterial activity.


INTRODUCTION
Traditionally, the use of plants in treating diseases and ailments has its origin in the history of man as it dates back to antiquity (Ogunyemi, 1979;Grabley and Thiericke, 1999). The use of plant by different ethnic groups for medicinal purposes prevails among the Nigerian people; even in the developed countries, plant derived drugs may be of some importance. In the United States of America, 25% of drug prescribed in the community pharmacies from 1959 to 1980 contain plant extracts or active ingredients obtain from them (Fransworth, 1999). As pointed out by Baker et al. 1995, plants have contributed significantly in ensuring the health care of about 80% of the world"s population. Most of the drugs produced from plant products arise from the traditional use of the plants.
For instance, 74% of the drugs derived from plant products listed by WHO arise from studies that isolated the active ingredients that accounted for using the plant in traditional medicine (Fransworth, 1999). Long before now, reports of resistance by pathogenic microorganisms to many synthetic drugs has been alarming (Ozumba, 2003;Aibinu, 2004). The continued increase in strains of microorganisms having resistant to antibiotic result in the development of a more effective antibiotic such as the 3rd and 4th generations of cephalosporins by pharmaceutical industries (Odugbemi, 2006). Several reports showing the potency of plant extract against microorganisms have been published. From these reports, plants became the foundation for synthetic drugs (Evans et al., 2002). B. ferruginea belongs to the family Euphorbiaceae. It mostly grows as a shrub but under favourable condition can grow well reaching the size of a tree. The tree is about 6 to 15 m high, and 1.5 m in girth. The bark is dark grey, rough and often scaly (Rashid et al., 2000). Its common names are Kirni (Hausa), Marehi (Fulfulde), Ira lodan (Yoruba), Ola (Igbo); and KensangeAbia (Boki). It"s mostly found in the savannah, most especially the moisture regions that extend from Guinea to the Democratic Republic of the Congo and Angola.
B. ferruginea has diverse uses. Extract from the bark have been used in the coagulation of milk and lime juice to formulate a traditional gargle The bark extract has been used for the coagulation of milk and also lime juice for the formulation of a traditional gargle "egunefu" (Orafidiya et al., 1990). Reports have also indicated its usage in the treatment of water (Kolawole and Olayemi 2003) In Togo, the bark of the root is used I the remedy of intestinal disorders and for the treatment of skin diseases (De Bruyne et al., 1997). Some other reported activity of the bark extract are trypanocidal (Ekanem et al., 2008), antimicrobial (Adeoye et al., 1988), molluscidal , anti-inflammatory (Olajide et al., 1999). The bark, leaves and roots are ingredients of Yoruba (in Nigeria) infusions chiefly administered to children (Burkil, 1994).
A large proportion of the population of African countries still rely on the use of local herbs in the management of many ailments ranging from surgical to medical either infectious and non-infectious, with different degree of Luka et al. 55 success or claim of beneficial effects. The bark and the bright red infusion from it are commonly sold in Nigerian markets and shops for use as a mouth-wash and remedy for thrush in children. In Congo, a bark decoction is used for toothache and in the Ivory Coast for dysentery and diarrhoea or as a laxative (Gill, 1992). The bark is used as antidote against poison and arrow poison (Burkil, 1994). A leaf extract in saline solution is reported to produce a marked reduction of blood-sugar in laboratory rats and clinical trials have given a drop from 250 mg to the normal less than 120 mg after eight weeks of daily treatment (Iwu, 1980). A bark preparation is used for immunity against arrow poison and syphilis. Extract from the bark is mixed with the stem of Costus for the treatment of minor epilepsy (Akubue and Mittal 1982). Root and stem barks are used for skin disease and eruption. They are found to be rich in tannins and are used as chewing sticks/mouth washes (Burkil, 1994). The boiled root and stem bark (boiled water extract) have recorded positive action against gram positive bacteria; Sarcinalutea and Staphylococcus aureus (Malcolm and Sofowora 1969). Antimicrobial properties of stem bark of Bridelia ferruginea against facultative gram negative rods have been reported by Ndukwe et al., 2007. The activities of the methanol, petroleum ether and chloroform bark extracts of B. ferruginea against some potential pathogenic organisms have been extensively investigated Adeoye et al., 1988;Olajide et al., 1999). B. ferruginea had been found to contain various organic compounds according to Sofowora et al., 1982. The plant was presumed to contain alkaloids, tannins, terpenoids, glycosides, flavonoids, saponins, anthraquinones and steroids.
The occurrence of multiple antibiotics resistance developed by microorganisms against the development of resistance to both the old and newer drugs calls for active search for more effective as well as affordable antimicrobial agents. This problem has prompted tremendous effort to explore for more potent antimicrobial agents, especially of natural origin to counter the resistance.
Previous studies have shown that, B. ferruginea has tremendous potentials as important sources of remedy for certain bacterial infections and as a source of new compounds for antimicrobial drugs syntheses (Sofowora, 1982).The plants are the sleeping giants of pharmaceutical industry (Hostettmann and Hamburger 1991), may provide natural source of antimicrobial drugs that will provide novel compounds that may be employed in the management some infections caused by microorganism.
Herbal preparations of B. ferruginea, have been found to be useful in treating some conditions but the extent of use is often met with a major setback from the fact that little scientific evidence is available for many of such preparations. In addition, the phytochemical properties of this plant have been found to vary with the habitat or geographical locations (Hostettmann and Hamburger 1991). Therefore, there may be variations in the antibacterial effect of this plant from different part of Nigeria. This study was aimed at finding and establishing a scientific basis for the use of the plant in search of effective and affordable cure for certain bacterial infections. The specific objective is to determine the phytochemical contents and antibacterial activity of the ethanolic and aqueous extracts of the stem and root bark of B. ferruginea.

Collection of plant materials
Fresh stem and root bark of B. ferruginea were collected from the field between July and October, 2015 and were identified by a botanist from the Federal College of Forestry, Jos, Plateau State.

Preparation of extracts
The plant materials (fresh stem and root bark of B. ferruginea) were dried at room temperature for 14 days followed by further drying in the oven at 50°C for 5 days to enable it dry completely. The dried stem and root bark were then pounded using a pestle and mortar and the powder stored in an air-tight container. The ground stem and root-bark of the plant was extracted using water and ethanol. About 20 g each of powdered root and stem-bark was poured in 120 ml of ethanol and water in a conical flask and the content sealed properly to avoid evaporation. In each case, the solution was filtered using Whatmann No. 1 filter paper after allowing the mixture to stand overnight. The filtrates were evaporated to dryness in an evaporating dish in a water bath at 70°C. The extracts were then scrapped and stored in a sterile container.

Phytochemical screening
The extracts of the stem and root bark of B. feruginea were analysed for the presence of the following phytochemicals; alkaloids, saponins, tannins, anthraquinones, and flavonoids according to standard methods (Sofowora 1982;Ngbede et al., 2008).

Source of the test organisms
The bacteria used for this study consist of six (6) clinical isolates; Pseudomonas aeroginosa, P. mirabilis, Escherichia coli, S. aureus, Bacillus cereus and Stapylococcus epidermidis. These isolates were obtained from the laboratory stock of central diagnostic, National Veterinary Research Institute, Vom, Jos. The gram positive and gram negative control strains; E. coli (NCTC 10418) and S. aureus (NCTC 6571) were also obtained from the Institute"s Molecular Laboratory. The pure cultures of these isolates were prepared in nutrient broth in test tubes and kept in the refrigerator at 4°C, until needed for use.

Constitution of the stock concentration of the extracts
About 5 g each of the aqueous stem and root-bark extracts were dissolved in 50 ml of distilled water to constitute a stock solution of 100 mg/ml. In similar manner 5 g of the ethanolic stem and rootbark extracts were dissolved in 50 ml of Dimethyl Sulphuroxide (DMSO) to constitute a stock concentration of 100 mg/ml. DMSO was used for ethanolic extract because it does not dissolve completely in water as in the case of aqueous extracts.

Determination of minimum inhibitory concentration (MIC)
Tube dilution method described by Scott, 1989) was used. Exactly 8 tubes labelled 1 to 8 (2 sets) with each tube containing 4 ml of nutrient broth. 4 ml of the crude extracts in the stock concentration (100 mg/ml) was introduced to tube 1 and diluted using a double dilution to yield a concentration of 50, 25, 12.5, 6.25, 3.12 and 1.56 mg/ml. Tube 8 contain a standard antibiotic (ciprofloxacin) serving as a positive control. To the first set of tubes, 0.02 ml (200µl) of 24 h broth cultures of the test organisms was added after dilution to yield 0.5 McFarland standard (equivalent to 1.5×10 8 Cfu/ml) using a Nephelometric turbidity metre. The tubes were then incubated at 37°C for 24 h after which they were examined for microbial growth. The second set of tube was not inoculated and serves as a control to observe for change in turbidity by comparing with the first set.The MICs of the ethanolic and aqueous extract of the stem and rootbark for each test organism is the smallest concentration of such extract that is capable of inhibiting the growth of specific inoculum of the test organism evidenced by absence of turbidity in the particular tube.

Determination of the minimum bactericidal concentration
The Minimum Bactericidal Concentrations (MBCs) was determined by identifying the tubes that showed no growth (evident by absence of turbidity) during MIC determination. One loop full from each of these tubes selected was inoculated over the surface of nutrient agar in Petri-dish and incubated for 18 to 24 h at 37°C. Lowest concentration of the extract at which no growth was observed is taken as the MBC.

RESULTS
The phytochemical evaluation of the aqueous extracts of the root and stem-bark, reveals the presence of some phytochemicals. The stem-bark extract contains alkaloids, saponins, tannins, and flavonoids. While rootbark extract demonstrated the presence of saponins, tannins, alkaloids, flavonoids and anthraquinones ( Table  1).
The result of the phytochemical analysis of ethanolic extracts of the root and stem-bark of B. ferruginea is shown in Table 2. The analysis of the stem extract revealed the presence of saponins, tannins, flavonoids, alkaloids, while anthraquinones was not present. The analysis of the root-bark extract demonstrated an increased presence of saponins, tanins and alkaloids, while, flavonoids and anthraquinones were present in moderate amounts. Table 3 shows the MIC and MBC of aqueous extract of the stem and root-bark of B. ferruginea. For stem-bark extract, amongst the gram positive bacteria tested, S. aureus (NCTC 6571) and S. aureus had no MIC and   Table 5 shows the summary of the primary activity of the root-and stem-bark extract of both the aqueous and ethanolic extract of B. ferruginea against the selected bacteria. The result indicates that, aqueous root-bark extract had more activity than aqueous stem-bark extract. On the other hand, the root-bark ethanolic extract had more activity than the stem-bark ethanolic extract. On a general note, the ethanolic root-and stem-bark extract had more activity than the aqueous root-and stem-bark extract with gram positive bacteria been more susceptible than the gram negative bacteria

DISCUSSIONS
Phytochemical analysis of the aqueous and ethanolic extracts of the root and stem bark of the plant revealed the presence of saponins, tannins, alkaloids, flavonoids and anthraquinones in the root-bark, while the stem-bark also possesses all the phytochemicals analysed, except for anthraquinones (Table 1 and 2).The presence of these phytochemicals reveals that, the extract of the plant is possibly active. The presence of these phytochemicals in the aqueous root-bark extract is in agreement with the findings of Adebayo and Ishola (2009) whereby all the phytochemical elements identified in this study were also found in their study. The absence of anthraquinones in the stem-bark extract, do not agree with the work done by Owoseni et al (2012) in which anthraquinones was present in the stem-bark extract.
The result from this study also revealed that there was no variation in terms of the active components in aqueous stem-and root-bark extract and that of ethanolic stem and root-bark however, there were variation in terms of the amount of the phytochemicals present in each of the extracts (Tables 1 and 2).There was an increase in the amount of saponins, tannins and flavonoids in ethanolic stem-bark extract, while ethanolic root-bark extract revealed an increase in the amount of saponins, tannins and alkaloids. In similar vein there was an increased extraction in the amount of the phytochemicals by ethanol when compared to aqueous agent. This finding is in line with Arya et al (2012) who observed higher yield using ethanol than other solvents such as petroleum ether, chloroform and water because ethanol is a more effective solvent in the extraction of bioactive molecules (Arya et al., 2012).
It has been discovered that the functional property of a plant relies upon the different secondary metabolites it  possesses such as: phenolics, terpenoids, or alkaloids (Murugan and Parimelazhagan 2014). Among the phytochemicals isolated from the plant, flavonoids have been proven to be of more significance because of its ability to assist the body in fighting diseases. Flavonoids act as potent antioxidants but depending on the structure of the molecule and the hydroxyl group in the chemical structure (Iqbal et al., 2015). Phenols, flavonoids and flavonols are polyphenolic compounds of plants which bring about substantial antioxidant activity and several biological activities including: anti-helmintic, analgesic, anti-inflammatory, anti-microbial and anti-allergic properties (Oyedemi et al., 2012;Alabri et al., 2014). The activity of these phytochemicals mainly results from their ability to form a large complex with extracellular protein and the cell walls of bacteria. Flavanoids can also alter the membranes of a bacterial cell (Tsuchiya et al., 1996).
The presence of saponins was indicated in B. ferruginea extracts which warrant the use of the plant in the management of inflammation. Saponins is known to prevent inflammation and is the main constituents of traditional medicinal plant and thus responsible for majority of the biological effects observed. This support reasons for using the plant in traditional medicine. Just et al., 1998 andIgbinosa et al., 2013 demonstrated the inhibitory effect of saponins on inflamed cells.
The presence of alkaloids is also observed in the extracts. A heterocyclic compound of nitrogen associated with a significant range of antimicrobial activity. Plants containing alkaloids are commonly used in traditional medicine due to their inhibitory effects against protozoa, bacteria and fungi (Kim et al., 2002). The common biological activity of alkaloids is toxicity against the cells of foreign organisms. It has the ability to accumulate in Table 5. Primary effects of the aqueous and ethanolic extracts of the stem-and root-bark of B. ferruginea against selected bacteria. cells due to the variation in membrane potentials and it is a very good DNA introducer (Iwasa et al., 2001) and effective against many microorganisms targeting the RNA polymerase, nucleic acid, RNA gyrase and topoisomerase (Yi et al., 2007). Plants that have extracts containing tannins are known to be used commonly in traditional medicine for treating haemorrhage, diarrhoea, and detoxification (Okwu and Emenike, 2006). The tannins content as observed in the study especially in the ethanolic extracts justify the use of the plant in traditional medicine for the treatment of diarrhoea. Anthraquinones have been found to have wide range of antibacterial (also anti-mycobacterial) property resulting in loss of function and inactivation of bacterial proteins, cell wall, polypeptides, adhesins and membrane bound organelles with resultant death of the bacterial cell (Kurek et al., 2011).

Bacteria Aqueous extracts Ethanolic extracts Stem-bark Root-bark Stem-bark Root-bark
The antibacterial activity of the plant extracts is tested on gram positive and gram negative bacteria. This is done by determining Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the aqueous and ethanolic extracts of the stem and rootbark of B. ferruginea (Tables 3 and 4). The presence of the phytochemicals is assumed to be responsible for the anti-inflammatory, antioxidant, antibacterial and cytotoxicity activity of the plants. From the results as shown in Table 3 and 4, the extracts have been found to be more active against gram-positive bacteria than gramnegative bacteria.
Among gram-positive bacteria tested, B. cereus was most susceptible to the plant extract having MIC and MBC of 25 and 50mg/ml, respectively for the stem-bark and root-bark ethanolic extract. For gram-negative bacteria, the plant extracts were most active against P. mirabilis with MIC and MBC of 50 and 100 mg/ml, respectively. The aqueous extract was most active against S. epidermidis with MIC and MBC of 50 and 100 mg/ml, respectively for stem-bark and 25 and 50 mg/ml, respectively for root-bark extract. The reason for the difference in sensitivity between gram negative and gram positive could be ascribed to the morphological differences between these microorganisms. As reported by Nostro et al., 2000;Nikaido and Vaara, (1985), gramnegative bacteria are characterized by an outer covering of phospholipidic membrane made up of lipopolysaccharide components, making the cell wall slightly impenetrable, whereas the gram-positive bacteria possessing only an outer peptidoglycan layer (not an effective permeability barriers) are more susceptible.
The level of resistance demonstrated by P. aeruginosa, E. coli and S. aureus is not strange since the resistances of these organisms to many antibacterial agents have been reported (Kunin, 1993). Also in another study by Dada-Adegbola et al, 2010, on "the activity of crude extract of B. ferruginea " against P. aeruginosa, E. coli and P. mirabilis showed similar results as obtained from this study.
The ethanolic extracts of both the stem-and root-bark demonstrated an increase in activity than that of aqueous stem-and root-bark extracts (Tables 3 and 4). The result from this study is consistent with the study by Owoseni et al. (2010), where the ethanolic extracts of leaves and stem-bark of B. ferruginea had an increase antibacterial activity than other extracting solvents used. Also, this result is in tandem with the findings of Olukemi et al. 1997, where ethanolic extract of stem-bark of Paekia filicoidea demonstrated ten-fold increase in activity to that of aqueous extracts. This is due to the concentration of active substances responsible for inhibiting the growth of microorganisms which is high in ethanolic extract than aqueous extract of both the root-and stem-bark of the plant. It could also be due to the fact that ethanol as extracting solvent is more effective in extracting the active compounds than aqueous solvent (Arya et al., 2012). The extracts from the root-bark were found to be more effective against the tested pathogens than the stem-bark for both the aqueous and ethanolic extracts (Table 3 and 4). Similar results were obtained by Adebayo and Ishola (2009) and Owoseni et al. (2012), where the root-bark ethanolic extract had higher activity than stem-bark extract against tested pathogens. The presence of anthraquinones in the roots of both the ethanolic and aqueous extract as against its absence in the stem extract could have been an attribute to its higher antibacterial activity as anthraquinones has been reported to have a wide range of antimicrobial activity against pathogenic organisms.
The primary effects of the aqueous and ethanolic extracts of the root-and stem-bark of the plant against selected bacteria as shown in Table 5 revealed that the antimicrobial activity of the plant is concentration dependent. Within the range of concentrations used for the study, the plant extracts were found to have the primary effect of being bacteriostatic or bactericidal, depending on the concentration against the test organisms. Only the stem-bark aqueous extract showed no primary effect on the control strains, S. aureus, E. coli, and P. aeruginosa. According to Oboh and Abulu (1997) antimicrobial activity is a function of the active ingredient having effect on the target organism.

Conclusion
Findings from this study confirmed the presence of phytochemicals such as saponins, tannins, alkaloids, flavonoids and anthraquinones which has revealed that the plant is of pharmacological importance going by the ability of these phytochemicals to elicit antibacterial activity.This study has discovered the great potential in B. ferruginea as one of the medicinal plant in serving as an alternative in treating human infection with less cost and toxicity.