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 (3-6). 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.
Declarations
Acknowledgment
The authors would like to thank the Department of Science Laboratory Technology, Adamawa State Polytechnic Yola, for their institutional support.
Conflict of Interest
The authors declare no conflicting interest.
Data Availability
You can access the Supplemental Table 1 on the ETFLIN server via the following link:
https://etflin.com/file/document/202305270733301064215839.xlsx
Ethics Statement
Not applicable.
Funding Information
Not applicable.
References
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, 9-11). 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 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.
Experimental Section
Materials
Collection of Plant Sample
AL was
obtained from the Girei Local Government area, Adamawa State, Nigeria, followed
by its identification and authentication by in the Forestry Department of
Adamawa State Polytechnic, Yola where a voucher specimen was deposited
(ASP/FT/245) after authentication by a Forest Technologist. It was cleaned,
dried under shade, and ground to powder.
Reagents and Chemicals
Methanol, chloroform, ethyl acetate, butanol, and diethyl ether were
purchased from Xingtai
Dakun Technology Co., Ltd (China). Nutrient agar and Mueller-Hinton agar (Qingdao Bio-Technology Co., Ltd., China). Amoxicillin (Zimilat®, Nemel Pharmaceutical,
Nigeria). All other chemicals and reagents were of AnarlaR.
Methods
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 using Equation 1.
TotalMetabolite(
Equation 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 550C. 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 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 using Equation 1.
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.
Antibacterial Activity
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.
PubChem and RSCB IDs for compounds, inhibitors, and targets were documented. Table 1 lists compound and standard drug PubChem IDs, while Table 2 details enzyme targets with PDB ID, grid coordinates, and box size.
Table 1. List of compounds and standard drugs (inhibitors).
Compounds
Name
PubChem ID
Designation
5-Methyl-1H-pyrazole-3-carboxylic
acid
9822
Compound I
1 2
4-Benzenetriol
10787
Compound II
2-Methoxyhydroquinone
69988
Compound III
Maltol
8369
Compound IV
Methyl
14-methylpentadecanoate
21205
Compound V
5-Hydroxymethylfurfural
237332
Compound VI
1
2-Epoxyhexadecane
23741
Compound VII
2-(Tetradecyloxy) ethanol
16491
Compound VIII
Methyl palmitate
8181
Compound IX
Hexadecanal
984
Compound X
Standard Drugs
Sulfamethoxazole
5329
Compound INB1
Cefepime
5479537
Compound INB2
Methotrexate
126941
Compound INB3
Fosfomycin
446987
Compound INB4
Ciprofloxacin
2764
Compound INB5
Afabicin
72696796
Compound INB6
Table 2. List of target enzymes with grid coordinate and box size.
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 using PyRx 0.8 software via the vina wizard with the
exhaustiveness set 32. The ligand with lowest binding energy was
selected and saved as PDB for visualization using PyMOL 2.5.4 and 2D
visualization was done with LigPlot+ 2.2.8. The
profiler web server was utilized to visualize other binding interactions (47). The binding energy was used to determine
the inhibition constant (Ki) using the formula Ki = exp ∆G/RT, where ∆G is the
binding energy, R is the universal gas constant = 1.985 x 10-3 kcal-1
mol-1 k-1, and T is the temperature (298.15 K) (48).
Figure 1. Zone of inhibition of AL extract. Note: Values with * were significantly (p<0.05) lower than A.E while values with # were significantly (p<0.05) lower than A.S.
Figure 2. 2D and 3D interactions of DS with Sulfamethoxazole and Methyl palmitate. (A) 2D Sulfamethoxazole; (B) 2D Methyl palmitate; (C) 3D Sulfamethoxazole; and (D) 3D Methyl palmitate.
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 standard amoxicillin exhibited a ZI of 30 ±1.00 mm and 20 ±2.00 mm for E. coli and S. aureus respectively at the concentration of 50 mg/mL. The least inhibitory effect was observed to be 7.0 ±1.21 mm and 2.0 ±1.31 mm respectively for E.coli and S. aureus at 25 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. Table 4 also presents the findings of the MBC which is the lowest
concentration at which the bacterial isolates are neutralized by the (AL)
extract. At a concentration >100 mg/mL E. coli was
not neutralized while S. aureus was completely neutralized at 100 mg/mL.
Table 4. MIC and MBC of AL extract against E.coli and S. aureus.
Test
Test organism
Incubation time (Hours)
Concentration (mg/ml)
Remark
100
50
25
12.5
6.25
MIC
E. coli
24
−
−
−
−
+
12.5
S. aureus
24
−
−
−
−
−
≤ 6.25
MBC
E. coli
24
+
+
+
+
+
> 100
S. aureus
24
−
+
+
+
+
100
Supplemental Table 1
(Sheet 1) provides an overview of the docking interactions between
dihydropteroate synthase (DS) and various compounds, including the inhibitor
sulfamethoxazole (INB1). Among the compounds, Compound IX (methyl palmitate)
demonstrated the highest ranking based on binding affinity (BA) (-6.4 kcal/mol)
and inhibition constant (Ki) (20 µM), along with INB1. While both INB1 and
Compound IX shared similar BA and Ki values, INB1 exhibited a greater number of
hydrogen bond (HB) interactions (7). On the other hand, Compound VIII (2-(Tetradecyloxy)
ethanol) had the highest BA (-4.2 kcal/mol) and Ki (828 µM), indicating it as
the least favorable interaction with DS. Among the compounds, Compound II (1,
2, 4-Benzenetriol) displayed a specific cation interaction (CI) with Arg255,
which was the only such interaction observed. To visually illustrate the
docking interactions, Figure 2 presents both the 2D and 3D representations of
Compound IX and INB1, highlighting the amino acids involved in hydrophobic
interactions (HBI) and HB, with the corresponding distances measured in
angstrom units.
The molecular docking
interactions of penicillin-binding protein 2X (PBP 2X) with the compounds and
its inhibitor are presented in Supplemental Table 1 (Sheet 2). The best docking pose with the lowest BA (-7.9
kcal/mol) and Ki (2 µM) was
exhibited by INB2 (Cefepime) with 1 HB, 12 hydrophobic interactions (HBI).
Compound IX (methyl palmitate) had the lowest BA (-7.1 kcal/mol) and Ki (6 µM)
among the compounds while compound VII (1, 2-Epoxyhexadecane) had the highest
BA (-4.4 kcal/mol) and Ki (590 µM) with 2 HB and 7 HBI. Only compound I
(-Methyl-1H-pyrazole-3-carboxylic acid) demonstrated salt bridge interaction
among the compounds by interacting with Lys340. 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.
The docking interactions between dihydrofolate reductase (DS) and various compounds, along with its inhibitor, are presented in Supplemental Table 1 (Sheet 3). INB3 (Methotrexate) demonstrated the lowest BA (-9.7 kcal/mol) and Ki (7.6 × 10-2 µM), along with 9 hydrogen bond (HB) interactions, 12 hydrophobic interactions (HBI), 1 cation interaction (CI), and 2 salt bridge (SB) interactions, positioning it as the most favorable docking pose. Compound VIII (2-(Tetradecyloxy) ethanol) exhibited the lowest BA (-6.2 kcal/mol) and Ki (28 µM) among the compounds, featuring 3 HB interactions and 15 HBI interactions. Notably, only compounds V and IX formed salt bridges with Arg255 among the compounds. Figure 4 visualizes the 2D and 3D interactions of dihydrofolate reductase (DS), highlighting the similarities in amino acid interactions between compound VIII and INB3.
Figure 3. 2D and 3D interactions of PBP2X with Cefepime and Methyl palmitate. (A) 2D Cefepime; (B) 2D Methyl palmitate; (C) 3D Cefepime; and (D) 3D Methyl palmitate.
Figure 4. 2D and 3D interactions of DR with INB3 and compound VIII. (A) 2D Methotrexate; (B) 2D 2-(Tetradecyloxy) Ethanol; (C) 3D Methotrexate; and (D) 3D 2-(Tetradecyloxy) Ethanol.
Figure 5. 2D and 3D interactions of MurA with Fosfomycin and Methyl palmitate. (A) 2D Fosfomycin; (B) 2D Methyl palmitate; (C) 3D Fosfomycin; and (D) 3D Methyl palmitate.
Figure 6. 2D and 3D Interactions of Compound IV and INB6 with TopoIV: a) 2D INB5 (Ciprofloxacin); b) 2D Compound VI (5-Hydroxymethylfurfural); c) 3D INB5 (Ciprofloxacin); and d) 3D Compound VI (5-Hydroxymethylfurfural).
Figure 7. 2D and 3D interactions of FabI with Afabicin and Methyl palmitate. (A) 2D Afabicin; (B) 2D Methyl palmitate; (C) 3D Afabicin; and (D) Methyl palmitate.
The interactions between UDP-N-acetylglucosamine enolpyruvyl transferase (Mur A) and its inhibitor, along with the compounds demonstrating their binding affinity (BA) and inhibition constant (Ki), are presented in Supplemental Table 1 (Sheet 4). Among the compounds, Compound IX (methyl palmitate) displayed the lowest BA (-7.2 kcal/mol) and Ki (5 µM), surpassing all other compounds, including INB4 (Fosfomycin). Interestingly, all the other compounds exhibited lower BA and Ki values than INB4, with Compound VI showing the least favorable interaction, featuring a BA of -4.4 kcal/mol and Ki of 590 µM. Compounds IV and II also engaged in specific cation interactions with Arg93 and Arg333, respectively. Figure 5 illustrates the amino acids involved in the interactions between MurA and INB5, as well as Compound IX, highlighting the hydrogen bond (HB) and hydrophobic interaction (HBI) patterns, along with the corresponding HB distances measured in angstrom units.
Supplemental Table 1 (Sheet 5) provides an overview
of the docking interactions between topoisomerase IV (TopoIV) and its
inhibitor, as well as the compounds, showcasing their binding affinity (BA),
inhibition constant (Ki), hydrogen bond (HB) interactions, hydrophobic
interactions (HBI), and van der Waals interactions (VWI). INB5 (Ciprofloxacin)
displayed the lowest BA (-7.8 kcal/mol) and Ki (2 µM), accompanied by 3 HB
interactions, 7 HBI interactions, and a salt bridge linkage with Glu46. Among
the compounds, Compound VI (5-Hydroxymethylfurfural) and I
(5-Methyl-1H-pyrazole-3-carboxylic acid) demonstrated the lowest BA (-5
kcal/mol) and Ki (214 µM). However, Compound VI exhibited 4 HB interactions and
4 HBI interactions, while Compound III (2-Methoxyhydroquinone) showcased the
highest BA (-4.2 kcal/mol) and Ki (828 µM) with 1 HB interaction and HBI.
Figure 6 visually presents the 2D and 3D binding interactions of INB6 and
Compound VI, including the amino acids involved and the corresponding HB
distances.
Figure 7 provides a visual representation of the 2D and 3D interactions between INB6, Compound IX, and FabI, highlighting the HB interactions, HBI interactions, and the corresponding HB distances measured in angstrom units. The docking interactions between
Enoyl-acyl-carrier-protein Reductase (FabI) and various compounds, including
INB6 (Afabicin), are outlined in Supplemental Table 1 (Sheet 6). INB6
demonstrated the lowest binding affinity (BA) (-7.6 kcal/mol) and inhibition
constant (Ki) (3 µM), with 2 hydrogen bond (HB) interactions and 8 hydrophobic
interactions (HBI). Among the compounds, Compound IX (methyl palmitate)
exhibited the least BA (-5.8 kcal/mol) and Ki (3 µM), featuring 7 HBI
interactions without any HB interactions. On the other hand, Compound II (1, 2,
4-Benzenetriol) displayed the highest BA (-3.7 kcal/mol) and Ki (1927 µM),
accompanied by a higher number of HB interactions compared to Compound IX.
Notably, none of the compounds, including the inhibitor, showed any cation
interaction (CI), salt bridge (SB), or pi-stacking (PS) interactions.
Table 5 summarizes the BA and Ki of the compounds
and inhibitors of the target enzymes. Among the compounds, III
(2-Methoxyhydroquinone) exhibited the least BA and Ki against 3 out of the 5
enzymes demonstrating a BA (-5.7 kcal/mol) against PBP2x, the least among all
the compounds across all the targets, thus, might be partly responsible for the
antibacterial action of AL.
Table 5. Summary of the Compounds with the least BA and Ki Against the Target Enzymes.
Enzymes
Compound
Standard Inhibitors
Designation
BA
(kcal/mol)
Ki
(µM)
Designation
BA
(kcal/mol)
Ki
(µM)
DS
IX
-6.4
20
INB1
-6.4
20
PBPX2
IX
-7.1
6
INB2
-7.9
2
DR
VIII
-6.2
28
INB3
-9.7
7.6 × 10-2
Mur A
IX
-7.2
5
INB4
-4.5
499
TopoIV
VI
-5
214
INB5
-7.8
2
FabI
IX
-5.8
55
INB6
7.6
3
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).
The
antibacterial activity of Anogeissus lieocarpus, against the test
bacteria, justified its traditional use as an ethnomedicinal plant by locals in
the study area. The presence of the identified phytochemicals may warrant the
plant's inhibitory function. Amoxicillin,
the positive control had the widest zone of inhibition. 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),
attributed to the presence of active phytochemical constituents which inhibits
bacterial growths. A bactericidal activity of AL extract was observed at 100
mg/ml for S. aureus but no impact on E. coli showing the relative
effect of concentrations, thus suggesting greater concentrations may be
necessary for E. coli as the present concentration maybe bacteriostatic.
Nonetheless, published results on the antibacterial effect of AL reported the
lethal effects of the extract on S. aureus (59) agreeing with the present study. The MIC values in this study
were different and lower than the MBC value obtained demonstrating that rather
than killing the organisms, the concentration used in the study was only able
to inhibit their growth. This is in tandem with the study carried out
previously (60). The lower MIC value
recorded for the study provides proof of the potent antibacterial properties of
A, consistent and in tandem with a previous study demonstrating a variety of
actions against a wide range of bacterial pathogens (61).
Dihydropteroate synthase (DS) catalyzes the synthesis of dihydropteroate via the conversion of
6-hydroxymethyl-dihydropterin 1′-diphosphate and 4-aminobenzoate yielding
dihydropteroate and inorganic pyrophosphate. This enzyme act as a target for
antimicrobial drugs such as Sulfamethoxazole because folic acid is not
synthesized by humans who depend on preformed folic acid. Sulfamethoxazole is
an analog of para-aminobenzoic acid (PABA) acting by competitively binding to
the enzyme preventing the binding of PABA which is the substrate for folic acid
synthesis (62). Although compound IX
interacted with higher BA and Ki, both compounds interacted with a similar
amino acid including Arg255, Lys221, Thr62, and Phe190 with compound IX
exhibiting HBI with all the amino acids while INB1 exhibited HB with Thr62 and
Phe98. The hydrophobic nature of compound IX might contribute to the favorable
interaction of compound IX with the DS binding pocket compared to the other
compounds. Although both INB1 and compound IX displayed the same BA and Ki, the
superior HB demonstrated by INB1 might offer a stronger interaction with DS
binding pocket than compound IX and offers more stability for the complex
formed. The binding of compound IX to DS might disrupt the activity of the
enzyme preventing folic acid synthesis via competitive inhibition. Thus,
the antibacterial effect of the AL extract might be partly attributed to the
action of compound IX, though a further study of the binding site might be
required to justify this claim. The fungicidal effects of compound IX and its
derivatives were previously reported (63).
Additionally, derivatives of compound II (1, 2, 4-Benzenetriol), were reported
to exert antibacterial activities depicting the compound as a precursor for
novel antibacterial compounds (64).
Penicillin-binding protein 2X (PBP 2X) is a class B PBP anchored to the
membrane participating in the final stage of peptidoglycan synthesis making
them targets of antibiotics specifically the β-lactam (65). β-lactam like INB2 (Cefepime) exhibits suicide inhibition
covalently binding to the active site of PBP 2X via acylation for an
extended period forming in active complex and preventing the catalytic action
of the enzyme subsequently blocking peptidoglycan synthesis and causing cell
lysis (66). Compound IX with the least BA
and Ki among the compounds exhibited a slightly higher BA and Ki compared to
INB2 interacting with similar amino acids though INB2 formed HB with Asn337.
The low BA and Ki of compound IX for PBP 2X might contribute to the
antibacterial effect of AL preventing the catalytic effect of the PBP 2X via
covalent bonding. Although Compound VI exhibited higher BA compared to
compound IX and INB2, it demonstrated a higher number of HB which might
translate to better stability. Thr550, Ser395, Ser337, Asn397, Glu552, Gln452,
and Lys340 were the identified amino acids interacting with INB2 and compound
IX participating in different binding interactions.
Dihydrofolate reductase (DR) catalyzes the formation of
5,6,7,8-tetrahydrofolic acid via the reduction of 7,8-dihydrofolate
through hydride transfer from the cofactor NADPH to the pterin ring yielding
5,6,7,8-tetrahydrofolic acid and NADP (67).
This is a critical reaction for maintaining tetrahydro folic acid level
required for nucleotide synthesis required for cell growth and proliferation,
thus a major target of antibacterial drugs considering the rapidly dividing
nature of bacterial cells (67).
Methotrexate exerts a broad-spectrum antibacterial effect against gram-positive
and some gram-negative by binding to DR thereby inhibiting folic acid synthesis
required for cell growth and proliferation (68).
In our study, INB3 (Methotrexate) exhibited a superior binding to DR than all
the compounds forming cation interactions with Phe31 and salt bridge with Asp27
and Lys32 in addition to the 9 HB and 12 HBI. This creates a better and more
stable interaction with the enzyme and inhibits its activity. Compound VIII
showed the least BA and Ki among the compounds that exhibited fewer HB but more
HBI than INB3. Some of the key amino acids identified participating in binding
interactions of DR with INB3 and compound VIII include Arg57, Asp27, Ile5, and
Ile94 which might be crucial for inhibiting the activity of DR, thus antibiotic
targets.
MurA enzyme is a transferase that catalyzes the transfer of the
enolypyruvate moiety of phosphoe-nolpyruvate (PEP) to UDP-n-acetylglucosamine
(UDP-GlcNAc) which marks the first step of the Mur pathway during bacteria cell
wall synthesis and being absent in eukaryotes makes them targets of many
antibiotics (69). Fosfomycin interferes
with bacterial cell wall synthesis by inhibiting the action of MurA via inactivating
the enzyme by covalently binding to the active site of the enzyme preventing
the early cytoplasmic stage of the cell wall synthesis (70). In the present study, INB4 exhibited higher BA and Ki against
MurA compared to all the compounds except compound VI. This might be attributed
to the non-interaction of fosfomycin with 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). In the present study, INB6 demonstrated the least BA and Ki
compared to all the other compounds displaying superior inhibitory effects
against fabI forming HB with Gly93 and Lys163 along with 8 HBI. This might
contribute to the stability of the complex formed with an extended binding
period. Among the compounds, IX exhibited the least BA and Ki without HB
interactions which might be attributed to the hydrophobic nature of the compound
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.
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.
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