J. Nig. Soc. Phys. Sci. 1 (2019) 6–11 Journal of the Nigerian Society of Physical Sciences Original Research Antimicrobial Assessment of Different Solvent Extracts of the Root-bark, Stem-bark and Leaves of Acacia ataxacantha Linn. O. S. Oguntoyea, A. M. Okoobohb, O. M. Belloc,d,∗, A. H. Usmana, S. S. Abdussalama, G. V. Awololad aDept. of Applied Chemistry, faculty of Science, Federal University Dutsin-Ma, Katsina State, Nigeria. bFederal College of Education, Pankshin Plateau State-Nigeria cDept. of Chemistry, Faculty of Science, University of Ilorin, Kwara State, Nigeria. dDept. of Industrial Chemistry, faculty of Science, University of Ilorin, Kwara State, Nigeria. Abstract Acacia ataxacantha DC. (Fabaceae) is one of the most important medicinal plant used traditionally in managing many ailments in Nigeria partic- ularly infectious diseases. In this study, the stem-bark, roots and leaves of A. ataxacantha were extracted using hexane, ethyl acetate, methanol and water (from non-polar to polar solvents). These extracts were initially screened to identify the secondary metabolites present. They were then examined against eight bacteria (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumo- niae, Streptococcus pneumonia, Proteus mirabilis, Salmonella typhi-murium) and seven fungi (Candida albicans, Penicillium spp, Aspergillus fumigatus, Aspergillus flavus, Trichophyton megninii, Trichophyton. rubrum, Salmonella typhi-murium). Disc diffusion method was employed to establish the antimicrobial activity of the plant parts. Serial dilution method was used to determine the minimum inhibitory concentration (MIC), minimum bactericidal (MBC) and fungicidal concentration (MFC). Methanol and hexane fractions of the root bark exhibited the highest inhibition zone ranging from 19-21 mm against the tested microorganism hence these were further assessed by determining their MIC, MBC and MFC. MIC value for the aqueous extract of the plant was 25 mg/cm3 against the fungi used while the methanol fraction was 15 mg/cm3 against the bacteria used. This work suggests that Acacia ataxacantha may possess compounds which can be template for acquiring novel drugs which could act against infectious diseases. Keywords: Acacia ataxacantha, infectious diseases, MIC, antimicrobial drugs Article History : Received: 13-03-2019 Received in revised form: 06-04-2019 Accepted for publication: 06-04-2019 Published: 09-04-2019 c©2019 Journal of the Nigerian Society of Physical Sciences All rights reserved. Communicated by: B. J. Falaye 1. Introduction Over a million and half of deaths associated with human diseases in the tropical countries are from infectious diseases. The cause of death and illness amongst inhabitants in develop- ing countries like West Africa can be traced to these infectious ∗Corresponding Author Tel. No: +2348062320327 Email address: obello@fudutsinma.edu.ng (O. M. Bello ) diseases [1]. Bacteria, viruses, parasites or fungi are some of these deadly microorganism, they are pathogen in nature and they are responsible for the alarming and high incidence of in- fectious diseases [2]. The recent discovery of developing resis- tance of pathogens to prevailing and current antibiotics is ob- vious [3]. There is a pressing demand to discover novel com- pounds with excellent antimicrobial activities because there has been a growing and disturbing increase in the occurrence of new and re-emerging infectious diseases [4, 5]. 6 Oguntoye et al. / J. Nig. Soc. Phys. Sci. 1 (2019) 6–11 7 Many bacterial species already have the genetic make-up to gain and transmit resistance against the current available antibi- otics. Many clinical challenges experienced around the world are as a result of the resistance posed by pathogens. The growth of this incidence is so alarming that, it has provided access to a new wave of widespread consumption of antibiotics [6-9]. The search for new antimicrobial agents is a serious burden due to the constant emergence of micro-organisms resistant to con- ventional antimicrobials, associated and adverse side effects of some antimicrobial drugs and the suddenly emergence of some uncommon infections [10, 11]. Pathogen resistance to currently used antibiotics increases death incidence, there is serious like- lihood of increase in patients visitation and stay in the clinics and hospitals [12]. Plant-based therapy has been used against array of diseases for generations hence looking to them for solution against mi- crobes may not be an exception [5,13]. Many secondary metabo- lites of excellent biological activities have been isolated from mother “Nature”. Nature wherein medicinal plants are found, provides a serious hope a better template in the discovery of new antimicrobial drugs [ 5, 14]. It is obvious from the litera- ture gathered that little work has been done on establishing the phytochemistry of Acacia ataxacantha. So far only five com- pounds have been isolated from the root-bark of this medicinal plant. Most biological evaluations were done on the root-bark, very few research was done on the stem bark while none on the leaves [15]. The medicinal plant is renowned traditionally for its antimicrobial uses, so there is a need to further explore, compare and justify using all the different parts i.e. stem bark, leaves and root-bark for antimicrobial use. This will serve as basis for further studies on the phytochemistry of this plant employing spectroscopic techniques to elucidate the active and non-active constituents in the fractions. 2. Materials and Methods 2.1. Plant material Different parts of A. ataxacantha were obtained from Jos, Nigeria in November, 2016. The plant was properly identified by the curator of the Herbarium, Department of Biological Sci- ence, University of Jos, Nigeria. 2.2. Extraction The root-barks, stem-barks and leaves were air-dried and grounded using wooden mortar and pestle. The powdered sam- ple was subjected to cold extraction (maceration) using hexane and then methanol. The crude methanol extract was suspended in water and washed with ethyl acetate, in that order, to yield water (WAF), methanol (MEF), ethyl acetate fraction (EAF) and hexane (HEF) respectively. 2.3. Phytochemical Screening Standard techniques of screening and detecting secondary metabolites in plants was used [16, 17]. The metabolites tested for were alkaloids, anthraquinones, cardiac glycosides, carbo- hydrates, flavonoids, saponins, steroids, tannins and terpenes. 2.4. Determination of Zone of Inhibition. The zone of inhibition of A. ataxacantha plant extracts were determined by using selected pathogenic micro-organisms ob- tained from the National Veterinary Research Institute, (NVRI) Vom, near Jos in Plateau State. Purity of the clinical isolates employed were verified and they were preserved in nutrient agar. The weight of the plant extracts (0.2 g) were taken, dimethyl sulphoxide (DMSO) (10 mls) was used to dissolve the plant extracts to have a fixed concentration of 20 mg/ml. This was the preliminary concentration of the extracts that was employed to test the antimicrobial properties of the plant extracts. The growth medium for the microbes was Mueller Hinton agar. The medium was prepared according to the Manufacturers guide- lines were followed in preparing the medium used. Further- more, zone of inhibition of A. ataxacantha plant extracts were evaluated according to the methods used by Lalnundanga et al. and Mathur [18, 19]. Then these plates of the medium were ob- served for the zone of inhibition of growth of the microbes, the diameter of the zones were taken and recorded in millimetres (mm). 2.5. Determination of Minimum Inhibitory Concentration (MIC) Resistance is a growing menace, MICs are often used by laboratories to check resistance, though it is also employed as a means to evaluate the antimicrobial activity (in vitro) of new agents [20]. The MIC of the methanol and water crude extracts of the root bark of A. ataxacantha was verified using Broth Di- lution method [21]. The broth labelled “Mueller Hinton Broth” was made by strictly following the manufacturers instructions. The MIC of the various solvents fractions of the parts of A. ataxacantha was determined using the modified agar well dif- fusion method [16, 18, 19, 22]. 2.6. Determination of Minimum Bactericidal Concentration (MBC). The minimum bactericidal concentrations (MBCs) of the water and methanol extracts were ascertained by selecting and sub-culturing the tubes that did not show growth during MIC determination, into fresh medium which contained no extract. Wire loop was used to transfer from these tubes, these were sub-cultured over the surface of extract free nutrient agar, in petri dishes. These were incubated for 24 h at 37 ◦C. The lowest extract concentration from which the organism did not recover and grow on the nutrient agar was documented as MBC. 2.7. Determination of the Minimum Fungicidal Concentration (MFC). Sabour and Dextrose Agar (SDA) plates were made fol- lowing the manufacturers instructions. The lowest dilution that did not show growth during MIC was inoculated onto the SDA plates. These plates were incubated at ambient temperature for 24 hours. The plates were then observed for growth. The lowest extract concentration from which the fungi did not recover and grow on the agar was noted as the minimum fungicidal concen- tration (MFC). The result is recorded in table 2.0. 7 Oguntoye et al. / J. Nig. Soc. Phys. Sci. 1 (2019) 6–11 8 2.8. Test Organisms The biological activities of the extracts were carried out at the National Veterinary Research Institute (NVRI), Vom, Plateau State, Nigeria. All the microorganisms used are of clinical strains from the same institute. The choice of these pathogens is based on their implication in human diseases such as typhoid, enteric fever, pneumonia, urinary tract infections, dysentery, wound infections and others. 3. Result and Discussion 3.1. Phytochemical Screening Phytochemical constituents extracts of A. ataxacantha are shown in Table 1. On the whole, coumarins, alkaloids, saponins, tannins, flavonoids, tannins, anthraquinones, cardiac glycosides and triterpenes were identified in all extracts. The ethyl acetate of the root-bark extract gave a poor result for most groups of secondary metabolites investigated. The phytochemical screen- ing reveals that flavonoids and carbohydrates are present in the various extracts. 3.2. Diameters of Zones of Inhibition The bacteria and fungi inhibition produced by A. ataxacan- tha extracts varied in relation to the type of extract and to the micro-organism strain used. Though most of the crude extracts exhibited varying degree of inhibition zones. Methanol and hexane extracts of the roots of A. ataxacantha had the high- est inhibition zone ranging from 10-19 mm against the tested bacteria used while the ethyl acetate extract of the stem bark and the hexane extract of the roots showed the highest zone of inhibition ranging 05 10 mm against the tested fungi. Implying that it is the more likely active fraction. The antibacterial ac- tivity of the exhibited by the roots extracts is good though from this study the antifungal activity is not too commendable. The antibacterial activity of the plant must be due to the phytochem- icals identified in the extracts. 3.3. MIC, MBC and MFCs Result 3.3.1. MIC This was carried out to determine the minimum concentra- tion of the extracts that can inhibit the growth of the microbes. The MIC results showed for only aqueous and methanol ex- tract of the root-bark for the plant, these were chosen because of their interesting activity by the zone of inhibition as Table 4 reveal. The extracts showed moderate activity against the bac- teria and fungi used. The water extract of the roots exhibited a moderate activity against the fungi with MIC of 25 mg/ml while the methanol extract of the same plant showed moder- ate activity against the bacterial with MIC ranging from 25-50 mg/ml. The former showed MIC of 25 mg/ml against P. aerug- inosa which a gram negative bacteria but the latter showed a better MIC against all the tested bacterial. 3.3.2. MBC and MFC Determination of MBC and MFC was done to check if mi- crobes evaluated were actually killed by the extracts or their growth was impeded. The results from MBC and MFC showed that none of the extract killed the bacteria after they were sub- cultured onto agar plate. That is when the results from MIC test tubes were sub-cultured, they still showed growth. This means none of the extract was bactericidal to the eight (8) bacteria employed for the study. In the same vein, the results showed no fungicidal effects on the three (3) fungi used in the study. In this study, the preliminary phytochemical screening tests were done for three different parts of the A. ataxacantha (leaves, stem bark and roots), each of the parts into four different polar- ities (water, methanol, ethyl acetate, hexane). The tests showed that alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, tannins and triterpenes were present in all extracts as shown in Table I. This screening may be useful in the discovery of the bioactive compounds and subsequently may lead to drug discovery and development. In antibacterial and antifungal studies carried out earlier, Akapa et al. (2015) in- vestigated the antibacterial and antifungal of the root-bark of A. ataxacantha using different polarities [23], it was reported that ethyl acetate fraction showed the most significant result with MIC of 2.5 mg/mL towards B. subtilis, E. coli, S. Typhi and K. pneumonia [24, 25]. The antifungal activity of stembark of A. ataxacantha extracts against six strains of Aspergillus was determined by Amoussa et al. [14] The ethyl acetate extract of the stem bark was reported to significantly inhibits the growth of mycelial and sporulation of Aspergillus strains. This study compares favourably with the literature where the stem bark extract (ethyl acetate fraction) of the plant was the most active against the fungi used. Looking at the results in table II and III, it was found that the stem bark extract (ethyl acetate fraction) showed the most interesting zone of inhibition (10 mm) against Candida albican in comparison with the control (0.5 mm), the same extract showed a better antibacterial activity against S. pneumoniae with an inhibition zone of 15 mm, which is better than to the control (10 mm). The ethyl acetate extract of the stem-bark showed an inhibi- tion zone of 5 mm against T. rubrum while the control showed no zone of inhibition. Looking at the results in the table III for antibacterial activity, it was found that the hexane and methanol extracts of the root bark showed the most interesting zone of inhibition (19 mm) against Pseudomonas aeruginosa in com- parison with the control “Cyprofloxacin” (19 mm), the hexane extract of the root-bark showed also an interesting antibacterial activity against S. pneumoniae with an inhibition zone which is equal to 15 mm, which is better than to the control (10 mm). Though the ethyl acetate extract of the leaves showed an in- hibition zone of 20 mm against S. aureus as same with the con- trol (20 mm). Figure 1 showed that methanol extract of the root- bark gave the highest zone of inhibition against S. typhi when compared with the other extracts. Ethyl acetate extract of the leaves of A. ataxacantha, methanol and ethyl acetate extracts of the stem-bark displayed similar zone of inhibition against the fungi used. Figure 2 revealed the antibacterial activities of 8 Oguntoye et al. / J. Nig. Soc. Phys. Sci. 1 (2019) 6–11 9 Table 1. The phytochemical screening of different parts of A. ataxacantha with solvents of different polarities. H2O : Water, MeOH : Methanol, E.A : Ethyl acetate, Hex : Hexane, +++ = Very Good, ++ = Good, + = Fair, - = Not present S/N Constituents Leaf Extract Stem bark Extract Root-bark Extracts H2O MeOH E.A Hex H2O MeOH E.A Hex H2O MeOH E.A Hex 1 Alkaloids + - - - - - - - - - - - 2 Saponins + + - - + +++ - - +++ ++ - + 3 Tannins ++ +++ ++ + ++ + - - - +++ - - 4 Flavonoids +++ +++ ++ + ++ ++ - - ++ ++ - - 5 Carbohydrates ++ ++ ++ + ++ +++ ++ + +++ ++ - +++ 6 Steroids + ++ ++ ++ - + ++ +++ - + + ++ 7 Anthraquinones - - - - ++ + - + ++ + 8 Cardiac glycosides - + + + - + + + - - - - 9 Terpenes - - ++ - - - - - - - + - % Yield 8.2 17 3 2 2.4 8.8 1 0.68 3 10.4 Table 2. Diameter of Zones of Inhibition of Fungi (mm). H20: Water, MeOH: Methanol, E.A: Ethyl acetate, Hex: Hexane, Values are means of three replicates (N = 3 ± S D). S/N Fungi Ctrl Leaf Extract Stem-bark Extract Root-bark Extracts H2O MeOH E.A Hex H2O MeOH E.A Hex H2O MeOH E.A Hex 1 Candida albican 0.5 0 0 0 08 0 0 10 0 0 06 0 0 2 Penicillium spp 0.8 0 0 0 0 0 0 0 0 0 0 0 05 3 A. flavus 10 0 05 08 05 03 03 08 05 03 0 0 05 4 A. fumigatus 12 0 05 05 0 05 05 05 05 05 0 0 05 5 T. megninii 0.3 0 0 0 0 0 0 0 0 0 0 0 05 6 T. rubrum 0 0 0 0 0 04 0 05 0 0 0 0 0 7 S. typhi 12 08 09 10 05 0 10 10 03 0 11 0 0 Table 3. Diameter of Zones of Inhibition of Bacteria (mm). H2O = water, MeOH = methanol, E.A = Ethyl acetate, Hex = Hexane, ketoconazole (An antifungal conventional drug), cyprofloxacin with (an antibacterial conventional drug). Values are means of three replicates (N = 3 ± S D). S/N Bacteria Ctrl Leaf Extract Stem-bark Extract Root-bark Extracts H2O MeOH E.A Hex H2O MeOH E.A Hex H2O MeOH E.A Hex 1 P. aeruginosa 19 0 0 0 0 0 0 0 0 0 19 0 19 2 E. coli 13 0 09 11 10 09 0 11 10 0 09 0 11 3 S. aureus 20 0 10 20 12 0 0 13 0 15 10 0 11 4 Bacillus spp 20 10 0 09 10 0 08 07 0 0 08 10 10 5 K. pneumoniae 30 10 15 15 10 10 10 10 05 11 10 0 10 6 S. pneumoniae 10 07 0 0 0 10 07 0 0 0 10 0 15 7 P. mirabilis 13 10 0 09 10 0 0 10 0 0 10 0 07 8 S. typhi 15 10 11 10 10 0 10 10 09 0 10 0 11 the various parts of this medicinal plant. Ethyl acetate extract of the leaves gave the highest zone of inhibition against S. aureus 9 Oguntoye et al. / J. Nig. Soc. Phys. Sci. 1 (2019) 6–11 10 Table 4. MIC result of the crude methanol and water extracts of the root-bark of Acacia ataxacantha. MER: methanol extract of the root, WER: water extract of the root, values are means of three replicates (N = 3 ± S D). S/N Test Organism Bacteria MER (mg/ml) WER (mg/ml) Control +ve Control - ve 1 Pseudomonas aeruginosa 200 25 + - 2 Escherichia coli 50 400 + - 3 Staphylococcus aureus 50 400 + - 4 Bacillus spp 100 400 + - 5 Klebsiella pneumoniae 100 400 + - 6 Streptococcus pneumonia 25 200 + - 7 Proteus mirabilis 400 400 + - 8 Citrobacter spp 50 100 + - Fungi 1 Candida abaccum 25 25 + - 2 Aspagirus flavus 100 25 + - 3 Aspagirus funigatus 100 25 + - while methanol extract of the root-bark showed a promising ac- tivity too. The presence the phytochemical compounds in these active extracts are responsible for the antimicrobial activity. The MIC results showed in Table IV are for aqueous and methanol extract of the root bark of the plant, these were cho- sen because of their interesting activity by the zone of inhibi- tion. The MIC values for methanol extract of the root was better than the aqueous extracts for bacteria while aqueous extract was better for the fungi tested. Aqueous extract showed the MIC values of 25 mg/ml against the fungi i.e. Candida abaccum, Aspagirus flavus, Aspagirus funigatus while methanol extract displayed a MIC value of 25 mg/ml against only Candida abac- cum and displayed MIC values of 100 mg/ml and 200 mg/ml against both Aspagirus flavus and Aspagirus funigatus respec- tively. The methanol extract of the root exhibited a moderate ac- tivity against Streptococcus pneumonia with MIC of 25 mg/ml while the aqueous extract of the same plant showed a very low activity against the same bacterial with MIC ranging from 200 mg/ml. MIC values for the aqueous extract are relatively low compared to methanol extract. This confirm the reported work of Akapa et al. [23] that methanol and chloroform fractions of root-bark had MIC values of 5 mg/ml and petroleum ether had 10 mg/ml for all the test organisms [23], though ethyl acetate gave the most significant result with 2.5 mg/ml for B. subtilis, E. coli, S. typhi and K. pneumonia [23, 24]. The result for MBC and MFC tests for both the methanol and water extracts on bac- teria and fungi are not significant. 4. Conclusion This study on the antimicrobial activity of different frac- tions of the roots, stembark and leaves of Acacia ataxacantha Figure 1. Antifungal activities of different parts of A. ataxacantha Figure 2. Antibacterial activity of different parts of A. ataxacantha 10 Oguntoye et al. / J. Nig. Soc. Phys. Sci. 1 (2019) 6–11 11 Linn were carried out employing disc diffusion method. The extracts were examined against eight bacteria (which consist of both + Gram and Gram bacteria) (P. aeruginosa, E. coli, S. au- reus, Bacillus spp, K. pneumonia, S. pneumonia, P. mirabilis, S. typhi ) and seven fungi (C. albican Penicillium spp, A. flavus, A. fumigatus, T. megninii, T. rubrum, S. typhi). Within the use of traditional Nigeria medicine, our studies are in agreement with the reputed potency of parts of A. ataxacantha against com- mon bacterial and fungal complaints. 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