Phytochemical Analysis, In-vitro , and In-silico Antibacterial Activity of Stembark Extract of Anogeissus leiocarpus Guill and Perr

: Bacterial infections subsequently leading to antibiotic resistance has been a leading cause of mortality and morbidity worldwide especially in developing countries with high poverty rate and poor healthcare system. Thus, prompting the prospect in alternative therapy such as medicinal plants. In the present study, we evaluated the antibacterial action of stem bark extract of Anogeissus leiocarpus (AL) Guill and Perr. as applied in folkloric medicine for antibacterial purposes. The phytochemicals present in the plant extract were identified and quantified, followed by the determination of the antibacterial effects of the extract against Escherichia coli and Staphylococcus aureus. Molecular docking study was carried out to ascertain the inhibitory effects of compounds from AL against bacterial enzymes. Alkaloids (7.17% ±0.60), saponins (11.33% ±3.18), and flavonoids (31.01% ±4.04) were detected. A maximum ZI was observed for E. coli compared to S. aureus at the highest extract concentration (100 mg/mL) with amoxicillin having superior ZI at 50 mg/mL concentration. The MIC against E. coli and S. aureus were 12.5 mg/mL and ≤ 6.25 mg/mL respectively while the MBC was>100 mg/mL and 100 mg/mL respectively. Among the identified compounds, IX exhibited the least binding affinity (BA) (7.2 kcal/mol) and inhibition constant (Ki) (5 µM) against UDP-N-acetylglucosamine Enolpyruvyl Transferase (Mur A) compared to all the other targets. AL demonstrated antibacterial activity evidenced by the bacterial growth inhibition, bactericidal potential, and in-silico study revealing high affinity of the bacterial enzymes for the identified compounds, thereby supporting the acclaimed antibacterial use of the plant in folkloric medicine.


Introduction
Bacterial infections (BI) coupled with antibiotic resistance (ABR) have been identified as the culprit leading to the death and morbidity of millions around the world, especially in places where there are a lot of challenges in the healthcare system such as developing countries (1). The menace of BI and ABR is expected to rise by 2050 with projected mortalities of up to 4 million worldwide (2, 3). In developing countries, poverty, poor access to modern healthcare facilities, and low government spending on healthcare might also contribute to the problems of BI and ABR (3)(4)(5)(6). There are several antibacterial drugs available in the market but as earlier stated, poverty plays a role as these drugs are often expensive and unaffordable. Additionally, these drugs are unavailable to people living in rural areas and often associated with side effects. Furthermore, the quality of these drugs is questionable which may or may not contribute to the antibacterial resistance of the drugs. Thus, rural communities are forced to prospect for local sources of drugs to achieve therapeutic goals.
E. coli are leading cause of many bacterial infections in both humans and animals with notable infections including urinary tract infections, septicemia and enteritis (7). Additionally, neonatal meningitis is also caused by E. coli while in farm animals, diarrhea has been associated with E. coli. The antibiotics resistance E. coli to major classes of antibiotics such as β-lactams, quinolones, aminoglycosides third-and fourth-generation cephalosporins and monobactams attributed to its outer membrane barrier further complicating treatment (7). S. aureus are associated with different human infections notably bacteraemia, infective endocarditis, skin and soft tissue infections osteomyelitis, septic arthritis, prosthetic device infections, pulmonary infections gastroenteritis, meningitis, and urinary tract infections (8). The type and duration of the treatment depends on the type of infection, however, emergence of antibiotic resistance by this organism further complicates treatment (8). E. coli and S. aureus are two of the frequently encountered bacterial infections in humans with their treatment complicated by antibiotic resistance leading to prospects into alternative therapies such medicinal plants especially in developing countries.
Medicinal plants provide plant-based drugs which are stipulated as an alternative to antibacterial drugs, notably in low-income countries due to the availability, safety, and efficacy evidenced by the traditional use of the plants in traditional medicine (6,(9)(10)(11). The use of plant-based sources of drugs is often attributed to their phytoconstituents produced by plants for several purposes such as defense against pathogens other than growth and reproductive functions (12). The pharmacological actions of these plants are due to their individual and synergistic mode of action via restoration of normal body function by allowing healing to take place (13,14). Medicinal plants exhibit several pharmacological effects including anti-inflammatory (15), and antimicrobial (16) effects via different mechanisms. The common antibacterial activity of medicinal plant extract is through the disruption bacterial of membrane functions, metabolic pathways, DNA and protein synthesis, and cell wall synthesis with synergistic mechanisms attributed to the inhibition of efflux pumps (16).
The pharmacological properties of the medicinal plants are attributed to the phytochemical composition including alkaloids, flavonoids, and saponins. Alkaloids exerts antibacterial effects via different mechanisms of action targeting different parts of bacteria and destroying its integrity. In a previous study, the alkaloids squalamine was reported to exert 16 to 32 times antibacterial effect than ciprofloxacin against Gram-negative pathogens (17). Indole-containing alkaloids were reported to exhibit antibacterial effects by inhibiting efflux pumps, the biofilm, filamentous temperature-sensitive protein Z, and methicillin-resistant Staphylococcus aureus pyruvate kinase (18).
Furthermore, alkaloids were postulated to be novel sources of antibacterial therapeutic (19). Flavonoids are also attributed with antibacterial activities where in some cases exhibiting more potential than standard drugs against multi-drug resistant pathogens including Gram-negative and Gram-positive bacteria (20). (−)-Epigallocatechin was reported to exhibit antibacterial effect via DNA synthesis inhibition in Proteus vulgaris and RNA synthesis in S. aureus. Saponins were also reported to inhibit S. aureus in dose dependent manner with minimal MIC and MBC values. Specifically, quinoa saponins disrupted cell wall synthesis and degraded cytoplasmic and protein membranes leading to loss of cellular integrity (21).
Ethnobotanical surveys identified A. leiocarpus (AL) as a plant used in the traditional management of infections and diseases. The plant is often utilized as decoctions prepared by aqueous macerations taken orally to overcome an infection (22). In other cases, the plant parts are ground to powder and applied to external wounds to prevent infection to allow the healing process to take place (23). In experimental studies, AL was reported to exhibit antihyperglycemic (24), antioxidant (24), antihyperlipidemic (24), and antimicrobial effects (25) through different modes of action attributed to its phytoconstituents including alkaloids, glycosides, and flavonoids. However, there are limited studies revealing the potential mechanisms of action Sciences of Pharmacy Dahiru MM (2023) -10.58920/sciphar02030024 https://etflin.com/sciphar of the compounds present in the plants. Moreover, phytochemicals exhibit antibacterial effect through individual or synergistic mechanism of action targeting different molecules, proteins, and enzymes to exert their therapeutic effects which might counter antibiotic resistance (26,27). Therefore, this study aimed to identify and quantitate the phytoconstituents of methanol stembark extract of A. leiocarpus and establish the antibacterial activity along with the potential mechanism of action in-silico as they are applied in folkloric medicine for antibacterial purposes.

Phytochemicals Extraction and Analysis
The phytochemical extraction was done via 48 h maceration of 300 g of stem bark AL in 1 L of 70% v/v methanol, then filtered and dried over reduced pressure (28). The phytochemicals in methanol stembark extract of AL (MSEA) were detected by the standard method described previously as follow:

Alkaloids
The estimation of total alkaloids was done as previously described (29). Briefly, 0.5 g of the extract was weighed into a conical flask containing 10 mL of 10 % ammonium hydroxide to convert alkaloidal salts into the free base; the mixture was stirred and allowed to stand for 4 h before filtering. The filtrate was evaporated to one-quarter of its original volume on a water bath and concentrated ammonium hydroxide solution was added dropwise to the mixture to precipitate the alkaloids. The precipitate was filtered using a weighed filter paper and washed with 10% ammonium hydroxide solution. The precipitate was dried with the filter paper in an oven at 60°C for 30 minutes and then reweighed and calculated thus; % Total Alkaloids = weight of residue weight of sample × 100 ( . 1) Where weight of residue = weight of the dried precipitate, weight of sample = weight of the extract taken earlier.

Saponins
Saponins were quantified by previously described methods (30). Exactly 0.5 g extract was introduced into a conical flask and 10 mL of 20% aqueous ethanol was added. The sample was heated over a water bath for 1 h with continuous stirring at about 55 0 C. The concentrate was transferred into a 250 mL separator funnel and 5 mL of diethyl ether was added and shaken vigorously. The aqueous layer was recovered and the ether Sciences of Pharmacy Dahiru MM (2023) -10.58920/sciphar02030024 https://etflin.com/sciphar layer was discarded. About 10 mL of n-butanol was then added followed by the addition of 2 mL of 5% aqueous NaCl. The remaining solution was heated over a water bath. After evaporation, the sample was dried in the oven to a constant weight and calculated as follows % Total Saponins = Weight of residue weight of sample × 100 ( . 2) Where weight of residue = weight of the dried residue, weight of sample = weight of the extract taken earlier.

Flavonoids
Flavonoids were estimated by the previously described method (29). Briefly, 0.5g of the extract was mixed with 10 mL of 80% aqueous methanol. The whole solution was filtered through the Whatman filter paper. The filtrate was transferred to a pre-weighed crucible and evaporated into dryness over a water bath and weighed.
% Total Flavonoids = weight of residue weight of sample × 100 ( . 3) Where weight of residue = weight of the dried filtrate, weight of sample = weight of the extract taken earlier.

Bacterial Isolates Collection
The bacteria isolates were obtained from the microbiology laboratory of Modibbo Adama University Teaching Hospital, Yola, Nigeria which were subjected to characterization as previously described (31).
Biochemical tests were carried out to ascertain isolated identity via the standard method (32,33), followed by growing on nutrient agar and subsequent storage at 4 °C.

McFarland Standard (MS)
MS preparation was done by mixing 9.95 mL of 1% H2SO4 and 0.05 mL of 1.17% BaCl forming a precipitate that acted as a 0.5 MS turbidity for the isolates (33).

Inoculum Standardization
Inoculum was standardized by culturing on nutrient agar and incubated at 37 °C overnight followed by transferring the formed colonies to test tubes with 5 mL of 0.9% normal saline adjusted to the turbidity of the MS (34).

Zone of Inhibition (ZI)
To ascertain the antibacterial activity of AL, a slightly modified agar diffusion technique was applied (35). The isolates were inoculated on a solidified Mueller-Hinton (MH) agar, followed by the addition of 0.2 mL extract at varied concentrations and added to five wells with the sixth acting as a positive control containing amoxicillin at 50 mg/mL. The mixture was incubated overnight at 37 °C. The antibacterial effect of the extract was expressed by the diameter of the ZI in mm.

Minimum Inhibitory Concentration (MIC)
The MIC was evaluated according to the protocols of the National Committee for Clinical Laboratory Standards (NCCLS) (36). One milliliter of extract was dispensed into 5 mL of MH broth containing test tubes and mixed to which 0.1 mL of the isolate broths were added and incubated overnight at 37 °C. The minimum concentration at which the bacterial growth was completely inhibited was defined as the MIC of the extracts.

Bactericidal Concentration (MBC)
Further evaluation of MBC was done by subculturing the test tube without visible growth in the MIC and incubating overnight at 37 °C. The least concentration without visible bacteria growth defined the MBC (37).

Molecular Docking
The compounds used for the in-silico study were collected from our previous study (38) were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov) along with the target inhibitors while the antibacterial targets were downloaded from the RSCB protein data bank (https://www.rcsb.org) in PDB format.
The PubChem and RSCB IDs of compounds, inhibitors, and targets were recorded. The list of the compounds and standard drugs (inhibitors) with their PubChem ID is provided in Table 1 while the targets enzymes and their PDB ID with grid coordinate and box size are provided in Table 2. The protein/receptor targets used for the present study were selected because they are targets of different antibacterial drugs (the roles of the proteins can be seen in the discussion section). The protein targets were downloaded already docked with their inhibitors prior to preparation using AutoDock Tools. The amino acid residues interacting with the inhibitors were marked and subsequently selected while choosing grid coordinate and box size.

Preparation of the Compounds, Inhibitors, and Targets
The list of the compounds along with their PubChem ID is provided in Table 1 while the targets and inhibitors with their PDB ID and PubChem ID respectively are provided in Table 2. The compounds and inhibitors were downloaded and converted to PDB format with Openbabel software version 3.1.1 (45). The targets downloaded in PDB format were prepared using AutoDock Tools, removing water molecules and hetero atoms 1.5.7 (46) and saved in PDB format to allow for proper docking of the ligands (compounds and inhibitors) with the target. The compounds and inhibitors downloaded were further subjected to energy minimization using the PyRx 0.8 software before docking.

Docking Procedure
The virtual screening of all the compounds and inhibitors against all the the targets was carried out

Statistics
The values obtained were expressed as mean ± standard error of triplicate determinations' mean (± SEM) and evaluated with Statistical Package for the Social Sciences (SPSS) version 22 software. One-way analysis of variance was used to assess the differences among the groups means followed by the Tukey multiple comparison test at p<0.05.

Result
The phytochemical components identified in the AL extract were alkaloids, saponins, and flavonoids.
Alkaloids had the least concentration of 7.17 ± 0.60%, while saponins had a concentration of 11.33 ± 3.18%.
Flavonoids were quantified in the highest concentration of 31.01 ± 4.04%. Figure 1 shows the antibacterial effects demonstrated by the AL extract on the bacteria isolates revealed by the ZI. A maximum ZI of 13.5 ±1.21 mm and 9 ±1.02 mm was observed for E. coli and S. aureus respectively at 100 mg/mL concentration.  The inhibitory effects of AL extract are presented in Table 4. The MIC of the AL extract against E. coli and S. aureus were 12.5 mg/mL and ≤ 6.25 mg/mL respectively.     Figure 3 shows the amino acids involved in the interactions of compound II and INB2 with PBP 2X with accompanied HBIs including the HB distance in angstrom.   Compounds IV and II also engaged in specific cation interactions with Arg93 and Arg333, respectively. Figure   5 illustrates

Discussion
Phytochemicals which are secondary metabolites including alkaloids produced by plants were reported to exert antibacterial effects with broad-spectrum effects (17,19). The antibiotic-enhancing and anti-virulence activity of flavonoids was previously reported (17). Indole alkaloids isolated from Pseudomonas aeruginosa were reported to exhibit potent antimicrobial action towards gram-negative and positive bacteria (49). Saponin compounds isolated from Chenopodium quinoa demonstrated anti-bactericidal activity towards S. aureus, S. epidermidis, and B. cereus with the highest activity recorded against S. aureus (21). Saponins from Albizia adianthifolia exerted considerable antibacterial effects against multi-drug resistant gram-negative bacteria (50). Flavonoids exert an antibacterial effect via disruption of the cell wall, protein, nucleic acid synthesis, and energy metabolism (51). Additionally, the cell membrane was predicted to be the target of flavonoids via phospholipid bilayer damage and disruption of ATP synthesis (52). Flavonoid reported in our study was not detected in a previous study on the methanol partitioned extract of AL (53). Similarly in another study, flavonoids were absent (54). In another study, alkaloids were absent in the MSEA but saponins and flavonoids were observed (55). The variation in the detection of phytochemicals in the methanol AL extract might be attributed to the difference in extraction methods (56). Compared to the crude extracts, this antibiotic demonstrated higher activity. This is not astonishing because it is expected that standard antibiotics should exert superior activity due to their refined natured compared to crude extracts. The relatively thin peptidoglycan layer of gram-negative bacteria and an outer phospholipidic membrane contain lipopolysaccharide components that result in lipophilic solute impermeability for Gram-positive bacteria, outer peptidoglycan layers are thick thus, not an effective and excellent permeable barrier which increase susceptibility to the plant extract (57). The MIC spanned from 6.25 to 12.5 mg/mL which is lower than the values previously documented (54,58) the key active site residues (Cys117 and Ser118) even though none of the compounds also interacted with these amino acids. Compound IX (methyl palmitate) exhibited the lowest BA and Ki among all the compounds which might be attributed to the hydrophobic nature of the compounds contributing to its stable interactions with residues within binding pockets.
Topoisomerase IV (TopoIV) is critical in maintaining the viability and genetic stability of cells by unraveling the newly formed DNA during replication to allow for the daughter chromosome separation as both the replication and segregation occur concurrently during cell division (71). TopoIV serves as an ideal target for many antibiotics inhibiting cell division such as ciprofloxacin which acts via topoisomerase II and IV inhibition (72). Ciprofloxacin is a broad-spectrum antibiotic for gram-negative and positive bacteria, binding its microbial target with 100 times more affinity than the mammalian target (73). In our study, INB5 (ciprofloxacin) exhibited superior BA and Ki compared to all the compounds with additional SB formation with Glu46. Among the compounds, VI (5-Hydroxymethylfurfural) and I (5-Methyl-1H-pyrazole-3-carboxylic acid) exhibited superior BA and Ki with compound VI showing a more stable interaction with the enzyme with more HB. Compound VI interacted with similar residues with INB5 but formed more HB which might be translated to extended binding time and lasting effect on the enzyme than INB5. The binding of compound IV to TopoIV with stability might disrupt the activity of the enzyme with bactericidal effects. Compound IV was previously linked to antibacterial activities (74). In another study, the compound was attributed with antibacterial effects against Acinetobacter baumanni through inhibition of biofilm formation and suppression of virulence regulator genes (75).
The fabI gene encodes the fabI reductase enzyme, a rate-limiting enzyme in the FAS-II pathway and an NADH-dependent enzyme catalyzing the last reaction of each round of elongation during the reduction of an enoyl-acyl carrier protein, thus a broad-spectrum antibacterial target and development of novel antibiotics (76). Afabicin is a first-class antibiotic targeting the bacterial fatty acid synthesis pathway (FAS-II) inhibiting the action of enoyl-acyl carrier protein reductase (FabI) (77

Conclusion
The present study evaluated the antibacterial actions of AL for its acclaimed use in folkloric medicine.
AL demonstrated antibacterial activity evidenced by the bacterial growth inhibition and bactericidal potential displayed by the plant in-vitro which might be attributed to the presence of phytochemicals. Furthermore, the in-silico study scientifically justifies the use of the effectiveness of the plant in the treatment of bacterial infections as claimed in folkloric medicine.

Funding
Not applicable.