In-vitro Activity and Molecular Docking of Treculia africana Leaf and Stem Bark Extracts against Multiple Resistant Clinical Isolates

Original Article

In-vitro Activity and Molecular Docking of Treculia africana Leaf and Stem Bark Extracts against Multiple Resistant Clinical Isolates

*Corresponding author: Oludare Temitope Osuntokun, Department of Microbiology, Adekunle Ajasin University, Akungba Akoko, Nigeria; E-mail: osuntokun4m@yahoo.com

Abstract

          Treculia africana is a highly valued economic plant, as well as an important medicinal plant widely used in the traditional herbal medicine for the treatment of several ailments of both microbial and non-microbial origins. Therefore, this study investigated the in vitro activity and molecular docking of Treculia africana against some pathogenic microorganisms. Antimicrobial screening of the ethanolic and ethyl acetate extracts of T. africana against the microorganisms was done using agar well diffusion method. Escherichia coli and Staphylococcus aureus were most susceptible to the extracts with zone of inhibition of 22 mm and 18 mm respectively while Pseudomonas aeruginosa showed resistant to the extract. Phytochemical screening revealed the presence of saponin and glycosides as the major components present in T. africana. Anthraquinone glycoside and polyphenols were also present. Molecular docking results revealed some components of the plant to be more active compared to levofloxacin by inhibiting topoisomerase IV. The efficacy of this plant against clinical and multiple resistant isolates could be explored for further pharmaceutical use.

Keywords

In vitro Activity; Molecular Docking; Treculia africana; Multiple Resistant Clinical Isolates

Introduction

          Treculia africana is a multipurpose tree species commonly known as African breadfruit. It belongs to the family Moraceae and it grows in the forest zone, particularly the coastal swamp zone [1]. It is widely grown in Southern Nigeria for its seeds and it is known by various tribal names in the country. Such names include Afon (Yoruba), Barafuta (Hausa), Ize (Bini), Eyo (Igala), Ediang (Efik) and Ukwa (Igbo) [2]. Most popular of these tribal names is the Igbo tribal name which is Ukwa. The species is a large tree which grows up to 30m high and its flowers between October and February [3].

African breadfruit is a traditionally important edible fruit tree in Nigeria whose importance is due to the potential use of its seeds, leaves, timber, roots and bark. It is increasingly becoming commercially important in Southern Nigeria. Baiyeri et al [4] described it as an important natural resource which contributes significantly to the income and dietary intake of the poor. The seeds are used for cooking and are highly nutritious as pointed out by various authors including Okafor et al [5]. The seeds have an excellent polyvalent dietetic value whose biological value exceeds even that of soybeans. Treculia africana is currently included in the list of endangered species of Southern Nigeria and this is quite worrisome. As a matter of fact, the species is urgently in need of priority conservation measures [6]. Therefore, this study seeks to investigated the in vitro activity and molecular docking of Treculia Africana as a natural plant which has potential of treating infectious diseases and clinical problems against some pathogenic microorganisms.

Figure 1: Treculia africana tree.

Materials and Methods

Source and Collection of Plants Samples

Plant materials (roots and stem bark) of Treculia africana were collected from the fresh water swamp forest, from sea level up to 3000 m altitude at latitude 70 28’ 40” N and longitude 50 44’ 15” E in Akungba, Akoko South East Local Government Area of Ondo State, Nigeria.

Authentication and Preparation of Plant Samples

The plants were authenticated at the Department of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba Akoko, Ondo State, Nigeria. The leaves and stem bark of Treculia africana were collected fresh in the early hours in the Month of August, 2017. The identified stem barks and leaves (1 kg) were washed, cut into small pieces and dried at an ambient temperature (40-50 °C), to ensure the sample lose its moisture content. The stem bark was powdered, after which the sample was then macerated using electronic blender. The leaves were powdered and milled at the department of Microbiology, Adekunle Ajasin University, Akungba Akoko, Ondo State, Nigeria.

Extraction of Plant Materials and Standardization of Plants Extracts

The extraction solvents used were ethyl acetate and ethanol. The extraction of each plant was done according to the method of Osuntokun [7]. At aseptic condition, the extracts were reconstituted by adding 1 g of each extract to 2.5 ml of DMSO and 7.5 ml of sterile distilled water, making it 100 mg/ml. For each extract, 5 ml of distilled water is measured into four sterile bijou bottles. The serial concentration was prepared to get concentration of 50 mg/ml, 25 mg/ml, 12 mg/ml, and 6.25 mg/ml respectively [7].

Antimicrobial Screening of the Extracts

The agar well diffusion method of Abioye et al., 2004 was used. The dried extract was reconstituted with sterile distilled water and ethyl acetate accordingly to the following concentration; 100, 50, 25, 12.5, and 6.25 mg/ml. Overnight broth culture of the respective bacteria strains were adjusted to turbidity equivalent to 0.5 McFarland standard. The plates were inoculated and incubated at 37 oC for 24 hours for the bacterial isolates and 24 oC for 48 hours for the fungi isolates. The zones of inhibition were measured and antimicrobial activity was expressed as the zone of inhibition diameters (mm) produced by the plant extract [7].

Determination of Minimum Inhibitory Concentration

The MIC of the extracts against the test organisms was determined using the broth dilution method as described by (El Astal., 2005). The Minimum Bactericidal Concentration (MBC) was determined by sub-culturing the plates that does not show bacterial growth from the results obtained in MIC determination [7].

Qualitative Phytochemical Analysis of Treculia Africana

  1. Test for Reducing Sugars: One milliliter of the plant filtrate was mixed with Fehling A and Fehling B separately; a brown colour with Fehling B and a green colour with Fehling A indicate the presence of reducing sugars [8].
  2. Test for Alkanol: The method of Mallikharjuna et al [9] was used. The powdered samples were wet with half diluted NH4OH and lixiviated with EtOAc for 24 hrs. at room temperature, it was sprayed with Dragendorff’s reagent. Orange spot shows a positive result [9].
  3. Test for Anthraquinone (Borntrager’s test): 50 mg of extract was heated with 1ml 10% ferric chloride solution and 1 ml of concentrated hydrochloric acid, the extract was allowed to cool and filtered. The filtrate was shaken with equal amount of diethyl ether. Further extraction of the ether with strong ammonia was done. Pink or deep red coloration of aqueous layer [10].
  4. Test for Cardiac glycosides, saponin, steroid and Flavonoid: The test for cardiacglycosides, saponin, steroid and flavonoid were done accordingly using the method of Mallikharjuna et al [9] and Onwukaeme et al [11].
  5. Test for Phenol: The extract was spotted on a filter paper, a drop of phoshomolybdic acid reagent was added and expose to ammonia vapors. Blue coloration of the spot shows a positive result [10].
  6. Test for Tannin (Braemer’s test): 10% alcoholic ferric chloride was added to 2-3 ml of methanolic extract (1:1) Dark blue or greenish grey coloration of the solution [10,12].

Phytochemical Quantitative Analysis of Treculia Africana

  1. Estimation of saponins

About 20 grams each of dried plant samples were homogenised, 100 ml of 20 % aqueous and ethanol were added. The mixture was heated using a hot water bath at 55 OC, for 4 hours with continuous stirring, the mixture was filtered and the residue was re-extracted further with a 200 ml of 20% ethanol. The combined extracts were reduced to 40 ml over a water bath at 90 °C. The concentrate was transferred and 20 ml of diethyl ether was added and then shaken vigorously in a 250 ml separatory funnel. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated three times. Then 60 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 10 m1 of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to a constant weight; the saponin content was calculated as percentage of the starting material (He, 2000) [13].

  1. Estimation of total flavonoid concentration

The concentration of flavonoids in the extract was estimated spectrophotometrically according to the procedure of Sun et al [14,15].

  1. Estimation of cardiac glucosides (Borntrager’s Test)

To 2 ml of filtrate hydrolysate, 3ml of ethyl acetate was added and shaken, ethyl acetate layer was separated and 10% ammonia solution was added to it. Formation of pink color indicated the presence of anthroquinone glycosides [16].

  1. Detection of alkaloid content

200 ml of 10% acetic acid in ethanol was added to five grams of the samples. The reaction mixture was covered and allowed to stand for 4 hours. These was filtered and the extract was concentrated to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop-wise to the extract until the precipitation was completed. The whole solution was allowed to settle and the precipitate was collected, washed with dilute ammonium hydroxide and then filtered; the residue being the alkaloid was dried and weighed to a constant mass [13,17].

  1. Estimation of phlobatannins

About 0.5 grams of each plant extracts was dissolved in distilled water and filtered. The filtrates were boiled in 2% HCl, Red precipitate showed the presence of phlobatannins. (Pharmacopeia, 2001).

  1. Estimation of total phenolic concentration

Estimation of total phenolic content was carried out using Folin-Ciocalteu’s phenol reagent reaction as reported by Singleton et al [18] Tsao [19].

  1. Determination of Tannins:

About 500 mg of the plant sample were weighed into a 50 ml plastic bottle. 50 ml of distilled water was added and shaken for 1 hour on a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to the marked level. Then, 5 ml of the filtrate was transferred into a test tube and mixed with 2 ml of 0.1 M FeCl in 0.1 M Hcl and 0.008 M potassium ferrocyanide. The absorbance was measured at 120 nm within 10 minutes. The tannins content was calculated using a standard curve of extract [20].

Molecular docking of T. Africana

In-silico approach was employed to study the interaction of plant phytochemicals from T. africana with three proteins; the Staphylococcus aureus topoisomerase iv, Salmonella typhi topoisomerase iv, and the yeast Candida albican 14α demethylase. (Walters et al., 1998) [21,22,23].

  1. Protein generation and preparation

The 3-dimensional crystallized structures of Staphylococcus aureus topoisomerase iv, Salmonella typhi topoisomerase iv and yeast Candida albican 14α demethylase were downloaded from the Protein Data Bank (PDB) repository (www.rcsb.org) with the PDB ID of 4URN and 5FSA with crystallographic resolutions of 2.30Aº and 2.86Aº respectively. The 3D structure of the Salmonella typhi topoisomerase iv (not found on the PDB repository) was retrieved by modelling the FASTA sequence of the protein gotten from the NCBI database (www.ncbi.nlm.nih.gov/protein/) using the swiss model server (www.swissmodel.expasy.org). The downloaded proteins were viewed with Schrödinger Maestro11.1. Proteins were prepared using Protein Preparation Wizard tool of the Schrödinger suite. The missing side-chains within the protein residues and the missing loops were filled using Prime (Schrödinger). The Co-crystallized water molecules, ions and cofactors were deleted, hydrogen atoms were added, and formal charges along with bond orders were assigned to the structures. The grid coordinate was generated around the co-crystallized ligand of the proteins with a grid box of 20Å×20Å×20Å.

  1. Ligand generation and preparation

A list of phytochemical constituents of T. africana was gotten from various literatures. The 2D structure of the ligands was retrieved from the NCBI Pubchem database. Under Schrödinger-Maestro tools, the respective 3D conformers of ligands were generated using the LigPrep. It also applies sophisticated rules to correct Lewis structures and to eliminate mistakes in ligands in order to reduce downstream computational errors (Wojciechowskiet al., 2005). Moreover, it optionally expands tautomeric and ionization states, ring conformations, and stereoisomers to produce broad chemical and structural diversity from a single input structure.

  1. Ligand Docking

This was carried out using GLIDE (Grid-based Ligand Docking with Energetics). Glide was run in rigid or flexible docking modes, and automatically generated conformations for each input ligand. The selection of the best pose was done on the interaction energy between the ligand and the protein as well as on the interactions the ligand shows with experimentally proved important residues(Schrödinger).Standard precision (SP) flexible ligand docking was carried out in Glide of Schrödinger-Maestro11.1 followed by the extra-precision (XP) mode which was used to combine a powerful sampling protocol with a custom scoring function designed to identify ligand poses that would be expected to have unfavorable energies, based on well-known principles of physical chemistry (Wojciechowskiet al., 2005).

Results

In this study, the medicinal plant Treculia africana leaves and stem bark were extracted in ethanol and ethyl acetate yield of dried grind powder. Ethanolic Treculia extract had higher inhibitory activity on Gram negative bacteria than in Gram positive bacteria. Escherichia coli was the most sensitive strain to the ethanolic leaf extract and exhibited the maximum zone of inhibition diameter of 22.0 mm at 100 mg/ml, while the lowest activity was demonstrated by leaf extract against Pseudomonas aeruginosa exhibiting the maximum zone of inhibition diameter of 10.0 mm at 100 mg/ml (Table 1). The ethanolic stem bark extract generally showed higher activity against the test organisms compared to the leaf extracts. That is, the stem bark extracts are more effective than the leaf extracts. This may be related to the fact that the stem bark was more developed and mature than the leaf which may contain fewer pigments and other phenolics which have been reported to interfere with the antimicrobial activity of the extract. The plant extracts evaluated showed a low activity against fungal isolates at the tested concentrations.     

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) has shown in this table reveals that, Pseudomonas aeruginosa had the highest MIC (100.0 mg/ml) and MBC (0.0 mg/ml), with Shigella sonnei and Staphylococcus aureus respectively. Pseudomonas aeruginosa, which was the least susceptible strain to the leaf extract, exhibited the most abundant zone of inhibition to the stem bark extract. This implies that it was the most susceptible strain to the stem bark extract with 20.0 mm diameter zone of inhibition at 100 mg/ml. The MIC and MBC values were generally lower for the leaf extracts against the test organisms compared to those of the stem bark extracts (Table 1). The highest MIC and MBC values of Staphylococcus aureus and Escherichia coli is an indication that either the plant extracts are less effective on some Gram-positive bacteria or that the organism has the potential of developing antibiotic resistance, while the low MIC and MBC values for other bacteria is an indication of the efficacy of the plant extract. The demonstration of broad spectrum of antibacterial activity by Treculia africana may help to discover newer classes of antimicrobial drugs [24]. The diameter zone of inhibiton, MIC and MBC values is an indication of a low efficacy of the plant extracts against the fungal isolates.

Table 1: Zones of inhibition of ethanol leaf and stem bark extracts of T. africana against selected clinical isolates.

Key:                                                                                                                           Unit = mm

L25– Leaf extract at 25 mg/ml                                           LMIC–Leaf extract (Minimum inhibitory conc)

SB25 – Stem bark extract at 25 mg/ml                            SBMIC– Stem bark (Minimum inhibitory conc)

L12.5– Leaf extract at 12.5 mg/ml                                  LMBC–Leaf (Minimum bactericidal conc)

SB12.5– Stem bark extract at 12.5 mg/ml                       SBMBCStem bark (Minimum bactericidal conc)

Table 2: In this study, the ethyl acetate Treculia africana extract had higher inhibitory activity on Gram negative bacteria than in Gram positive bacteria. Staphylococcus aureus was the most sensitive strain to the ethanolic leaf extract and exhibited the maximum zone of inhibition diameter of 18mm at 100 mg/ml, while the lowest activity was demonstrated by leaf extract against Pseudomonas aeruginosa exhibiting the maximum zone of inhibition diameter of 10 mm at 100 mg/ml. The ethyl acetate stem bark extract generally showed higher activity against the test organisms compared to the leaf extracts. That is, the stem bark extracts are more effective than the leaf extracts. This may be related to the fact that the stem bark was more developed and mature than the leaf which may contain fewer pigments and other phenolics which have been reported to interfere with the antimicrobial activity of the extract as discussed earlier. The plant extracts evaluated showed a low activity against fungal isolates at the tested concentrations.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) has shown in this table reveals that, Staphylococcus aureus had the highest MIC (25 mg/ml) and MBC (50 mg/ml), while the lowest MIC of 100 mg/ml was demonstrated by Escherichia coli, Shigella sonnei, Staphylococcus aureus and Candida albican respectively.

Table 2: Zones of inhibition of Ethyl acetate leaf and stem bark extracts of T. africana against selected clinical isolates.

Key:                                                                                                 Unit = mm

L100 Leaf extract at 100 mg/ml                              L25Leaf extract at 25 mg/ml

SB100Stem bark extract at 100 mg/ml                   SB25 – Stem bark extract at 25 mg/ml

L50 Leaf extract at 50 mg/ml                                  L12.5Leaf extract at 12.5 mg/ml

SB50 Stem bark extract at 50 mg/ml                        SB12.5Stem bark extract at 12.5 mg/ml

 

LMIC–Leaf extract (Minimum inhibitory conc)

SBMIC– Stem bark (Minimum inhibitory conc)

LMBC–Leaf (Minimum bactericidal conc)

SBMBC–Stem bark (Minimum bactericidal conc)

Table 3: The ethanolic stem bark extract of T. africana showed an effective activity in all multiple resistant bacteria strains in the solid agar well diffusion test. All bacterial strains tested were Gram negative bacteria thus; there was a high level of inhibitory activity. Ethanolic stem bark of Treculia extract had higher inhibitory activity on tested bacteria than the leaf extract. Enterobacter agglomerans compex and Enterobacter agglomerans were the most sensitive strain to the ethanolic stem bark extract and exhibited the maximum zone of inhibition diameter of 21 mm and 20 mm at 100 mg/ml respectively, while the lowest activity was demonstrated by leaf extract against Acinetobacter baumannii exhibiting the maximum zone of inhibition diameter of 10 mm at 100 mg/ml. The ethanolic leaf extract generally showed a lower activity against the test organisms compared to the stem bark extracts. This implies that, the stem bark extracts are more effective than the leaf extracts. Strenotrophomonas maltophilia and Acinetobacter baumannii showed a maximum zone of inhibition diameter of 10 mm and 8 mm at 100 mg/ml respectively.

Table 3: Zones of inhibition of Ethanol leaf and stem bark extracts of T.africana against multi drug resistant isolates.

Key:                                                                                                  Unit = mm

L100 – Leaf extract at 100 mg/ml                       L25– Leaf extract at 25 mg/ml

SB100– Stem bark extract at 100 mg/ml           SB25– Stem bark extract at 25 mg/ml

L50– Leaf extract at 50 mg/ml                         L12.5 -Leaf extract at 12.5 mg/ml

SB50– Stem bark extract at 50 mg/ml               SB12.5 –Stem bark extract at 12.5 mg/ml

Table 4: Ethylacetate stem bark Treculia extract had higher inhibitory activity on tested bacteria than the leaf extract. Enterobacter agglomerans and Acinetobacter baumannii were the most sensitive strain to the ethylacetate stem bark extract and exhibited the maximum zone of inhibition diameter of 25 mm and 22 mm at 100 mg/ml respectively, while the lowest activity was demonstrated by leaf extract against Morganella morganii exhibiting the maximum zone of inhibition diameter of 13mm at 100mg/ml. The ethyl acetate leaf extract generally showed a lower activity against the test organisms compared to the stem bark extracts. This also implies that; the stem bark extracts are more effective than the leaf extracts. Strenotrophomonas maltophilia and Acinetobacter baumannii showed a maximum zone of inhibition diameter of 15 mm and 9 mm at 100 mg/ml respectively. Enterobacter agglomeran, Morganella morganii and Enterobacter agglomeran complex showed 0.0 mm at 100 mg/ml, 50 mg/ml, 25 mg/ml and 12.5 mg/ml respectively.

Table 4: Zones of inhibition of Ethyl acetate leaf and stem bark extracts of T. africana against multi drug resistant isolates.

Key:                                                                                            Unit = mm

L100 – Leaf extract at 100 mg/ml                       L25– Leaf extract at 25 mg/ml

SB100– Stem bark extract at 100 mg/ml           SB25– Stem bark extract at 25 mg/ml

L50– Leaf extract at 50 mg/ml                          L12.5 -Leaf extract at 12.5 mg/ml

SB50– Stem bark extract at 50 mg/ml              SB12.5 –Stem bark extract at 12.5 mg/ml

Table 5: The phytochemical constituents of the plant extracts consisted of glycosides, alkaloids, anthraquinone, tannins, flavonoids, saponin, reducing sugars, steroids, and phenols. Phytochemical constituents such as anthraquinone, flavonoids, alkaloids and several other aromatic compounds are secondary metabolites of plants that serve as defense mechanisms against predation by many microorganisms, insects and herbivores (Lutterodt et al., 2005). Perhaps, this explains the antimicrobial potency of the leaf and stem bark extracts of Treculia africana.

The phytochemical screening with methanol showed a conspicuous absence of steroid, phenol and reducing sugar but presence of alkaloids, including saponin, glycosides, with anthraquinone glycosides and tannins in the leaf extract. Acetone extract reveals the presence of alkaloids, steroids, phenol, tannins and flavanoids. Anthraquinone was not indicated while Cardiac glycoside, saponnin and reducing sugars were negative. Dichloromethane and ethyl acetate extract reveals the presence of all phytochemicals except reducing sugars and anthraquinone. The stem bark methanolic, acetonic, dichloromethanic and ehtylacetate extract reveals all phytochemicals present except anthraquinone which was not indicated.

 

 

 

Table 5: Qualitative phytochemical analysis of Treculia africana using different solvents

 Key:     

A = Methanol                      B = Acetone                         C = Dichloromethane                         D = Ethyl acetate

Table 6: The quantitative phytochemical analysis result reveals the specific measured amount of constituents present in both plant extracts. It was observed from the result that the methanolic leaf and stem bark extract has a relatively different amount of phytochemicals present, ranging from 8.55-1.50 and 14.04-1.67 respectively. From the leaf extract Saponin has the most abundant phytochemical with 8.55 and others ranged from 6.00-1.00. It was observed from the stem bark extract that tannin has the most abundant phytochemical with 14.04 and others ranging from 13.00-1.00. The quantitative analysis of leaf and stem bark using acetone shows the compound presence ranging from 2.45-1.92 and 3.58-3.00 respectively. Saponin and alkaloids were observed to be the most abundant from the leaf extract with 2.45 and the flavonoid with 1.92. Alkaloid has the most abundant compounds from the stem bark extract with 3.58 and the least compound was found in tannin with 3.00. the results from the leaf and stem bark using dichloromethane and ethylacetate shows a slight difference in the amount of compounds present, while some phytochemicals has the same amount of compounds present as in cardiac glycoside with 3.12, tannin with 6.89, flavonoid with 2.56, saponnin with 2.12, tannin with 4.98 using leaf extract. From the stem bark extract, alkaloid has 0.93, and phenol with 4.72.

Table 6: Quantitative phytochemical analysis of Treculia africana using different solvents.                                      

            Key:A = Methanol                      B = Acetone

C = Dichloromethane                         D = Ethyl acetate

Table 7: shows that tricyclene-1 and terpinen-4-ol-1 which are phytochemicals present in Treculia africana are more active against Salmonella typhi by inhibiting the topoisomerase IV than levofloxacin, with docking scores of -2.267 and -1.625 respectively, while levofloxacin has a docking score of -1.557.

The result also shows that Terpinen-4-ol.sdf, Myrtenal.sdf, GAMMA-TERPINENE.sdf, ALPHA-TERPINENE.sdf, P-CYMENE.sdf, (+)-Sabinene.sdf have the docking score of -3.398, -3.293, -3.075, -3.05, -2.968, and -2.868 respectively. These phytochemicals are less active than levofloxacin which has the docking score of -3.436. This means that the phytochemicals which are less active against Staphylococcus aureus cannot inhibit the topoisomerase IV of Staphylococcus aureus.

The Candida albican docking result shows that coligand5fsa-1, alpha-Humulene-1, BETA-CARYOPHYLLENE-1, Elemol-1, Beta-Acoradiene-1, P-CYMENE-1, and Terpinen-4-ol-1 present in Treculia africana have docking scores of -9.835, -7.63, -7.544, -7.307, -6.78, -6.331, and -5.646 respectively, which is higher than the docking score of levofloxacin, which is -1.557. This means that coligand5fsa-1, alpha-Humulene-1, BETA-CARYOPHYLLENE-1, Elemol-1, Beta-Acoradiene-1, P-CYMENE-1, and Terpinen-4-ol-1 are more active against Candida albican than levofloxacin by inhibiting the DNA gyrase.

Table 7: shows the docking scores of the phytochemicals present in T. diversifolia plant against clinical isolates.

Discussion

The rising prevalence of antibiotics resistant pathogenic microorganisms in the last decades raises the demand for finding new alternative antimicrobial agents. This plant is used in traditional medicine in the treatment of skin diseases, gastrointestinal tract diseases and respiratory problems [25].

The result of antimicrobial susceptibility assay demonstrated a promising evidence for the antimicrobial effects of T. africana against bacterial and fungal isolates used in this study. The MIC and MBC values were generally lower for the root extracts against the test organisms compared to those of the stem bark extracts. The highest MIC and MBC values of Staphylococcus aureus is an indication that either the plant extracts are less effective on some gram-positive bacteria or that the organism has the potential of developing antibiotic resistance, while the low MIC and MBC values for other bacteria is an indication of the efficacy of the plant extract.

The ethanol stem-back extract of T. africana was found to exert an antimicrobial activity against the bacterial and fungal strains used in this study. All the strains tested were inhibited in the susceptibility test except P. aeruginosa which was not susceptible at a very low concentration because of its low antibiotic susceptibility which is attributable to a concerted action of multi-drug efflux pumps with chromosomally encoded antibiotics resistance genes and low permeability of the bacterial cellular envelopes. Mutations in DNA gyrase are also commonly associated with antibiotic resistance on P. aeruginosa. S. aureus has been reported to be highly resistant to ampicillin, cephalexin, methicillin and vancomycin; and is also resistant to gentamycin, rifampicin and chloramphenicol [25], (Donaldson and Gosbell, 2006). The reason for the differential sensitivity between Gram-positive and Gram-negative bacterial strains could not be ascribed to their morphological differences or adduced to their chemical compositions. Gram negative bacteria have an outer phospholipids membrane with the structural lipopolysaccharide components, which make their cell wall impenetrable to antimicrobial agents (Nikaido and Vaara, 2010), while the Gram-positive bacteria should be more susceptible having only an outer peptidoglycan, which is not effective permeability barrier [26]. In spite of the permeability difference, the ethanol extract of T. Africana exerted a broader spectrum of inhibitory activity on Gram-negative bacteria than on Gram-positive bacteria strain. The higher activity of the extract against P. aeruginosa, S. typhi, S. dysentriae and E. coli which are at times responsible for the pathogenesis of urinary and enteric infections provides a scientific evidence for the efficacy of T. africana in treating such infections [27].

Ethanolic Treculia extract had higher inhibitory activity on Gram negative bacteria than in Gram positive bacteria. Escherichia coli being the leading cause of urinary tract, ear, wound, and other infections in human was the most sensitive strain to the ethanolic leaf extract. The ethanolic stem bark extract generally showed higher activity against the test organisms compared to the leaf extracts.  This may be related to the fact that the stem bark was more developed and mature than the leaf which may contain fewer pigments and other phenolics which have been reported to interfere with the antimicrobial activity of the extract. The plant extracts evaluated showed a low activity against fungal isolates at the tested concentrations.     

The demonstration of broad spectrum of antibacterial activity by Treculia africana may help to discover newer classes of antimicrobial drugs [24]. The ethyl acetate stem bark extract generally showed higher activity against the test organisms compared to the leaf extracts.

The antimicrobial activity using ethanol stem bark extract of T. africana showed an effective activity in all multiple resistant bacteria strains in the solid agar well diffusion test. There was a high level of inhibitory activity on all Gram-negative bacteria.

Phytochemical screening helps to reveal the chemical nature of the constituents of the plant extract and the one that predominates over the others. It may also be used to search for bioactive lead agents that could be used in the partial synthesis of some useful drugs [28]. Saponins are characterized by their surface-active properties and they dissolve in water to form foamy solutions and because of surface activity some drugs containing saponins have a very long history of usage. Saponins have been implicated as a bioactive antibacterial agent of plants containing them [29,30]. The exhibited antibacterial properties of T. africana can be attributed to the presence of steroidal saponins and polyphenols in the bark. Polyphenols were earlier reported to have some antibacterial activities (Tomas Barberan et al., 2000) and might have complimented or potentiated the saponins in the antibacterial activities exhibited by T. africana bark extract.

The molecular docking results shows that tricyclene-1 and terpinen-4-ol-1 which are phytochemicals present in Treculia africana are more active against Salmonella typhi by inhibiting the topoisomerase IV than levofloxacin,

Characterization of the binding behavior has helped to predict the binding-conformation of the ligands present in T. africana to the targeted binding site of the test organisms. The strength and the signal produced by the plant extracts were also predicted.

Conclusion

This study investigated the effects of ethanolic and ethyl acetate leaf and stem back extract of Treculia africana on clinical isolates and multiple resistant organism. Due to the challenges associated with drug resistance, which have made scientists to research for effective and sustainable means of managing the problem. Plants have emerged as an alternative to synthetic antibiotics which is prone to reoccurring drug resistance. Therefore, demonstration of broad spectrum of antibacterial activity by Treculia africana may help to discover newer classes of antimicrobial drugs.

Recommendation
          This under-utilized plant Treculia africana need to be further studied to find out the bioactive natural compounds that would facilitate and open up new pharmacological discoveries that may lead to synthesis of a more potent drug with little or no adverse effect. Therefore, more work is recommended.

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