1 Introduction

Morinda lucida is a plant species that is widely distributed in the tropics, especially in West Africa. The plant has numerous medicinal properties. It is used traditionally in various communities for the treatment of different ailments [1]. One of the reported applications of M. lucida is the treatment of bacterial infections [2, 3]. In recent years, the use of plant extracts for the development of new antibacterial agents has been on the increase, particularly in the face of the increasing prevalence of antibiotic-resistant bacteria [4, 5]. Approximately 80% of people worldwide still rely on plant medicines for relief from illnesses, according to the World Health Organization (WHO). Since they are an essential source of physiologically active compounds, medicinal plants have a big impact on human health. [6,7,8]The study of M. lucida root extracts by in vitro and in silico antibacterial analysis is highly intriguing as it offers important information about the plant's potential for producing new antibacterial chemicals. Previous studies have reported the antibacterial potential of the leaves and stem bark of M. lucida against a range of bacterial strains and its use in traditional medicine [2, 5, 9,10,11]. The M. lucida leaf extracts have been found to have the potential to inhibit the growth of E. coli and Salmonella species, indicating its potential in the treatment of infections involving these bacteria [6]. Morinda lucida leaf aqueous extract has strong immunostimulatory and immunorestorative qualities, which may account for why it works so well for treating infections and immuno-inflammatory conditions [7]. The ethanol extract of M. lucida leaves possesses antiemetic properties, suggesting its potential for use in the development of new antiemetic drugs [8] and the leaves may have potential applications in anticoccidial therapy [12].

A different study discovered that the M. lucida leaf aqueous extract had the greatest inhibitory effect on two enzymes (α-Amylase and α-Glucosidase), and this activity might be because of phytochemicals like tannins, flavonoids, and saponins [13]. Importantly, research indicates that consuming M. lucida leaf extract has no negative effects on liver or kidney function, indicating that it might be a safe therapeutic herb [14]. According to Saganuwan et al., the kidney, lung, liver, heart, and spleen may have mild to moderate adverse effects at higher dosages. However, the aqueous extract of M. lucida is safe when administered for 28 days at test dosages of 16.26 and 162.65 mg/kg body weight. [15]The Staphylococcus aureus (a Gram-positive multidrug resistant [16, 17] facultative anaerobe bacteria typically causing a variety of clinical diseases [18]) and Pseudomonas aeruginosa (a ubiquitous facultatively anaerobic, toxin-producing gram-negative bacterium [19] associated with serious infections in immunocompromised hosts [20]). The GC–MS analysis of M. lucida was discussed by Adekunle et al., [21] indicating that the root of M. lucida is very rich in phytocompounds. The information regarding the phytochemical profile and biological functions of M. lucida roots has not yet been fully utilized. As a result, the study sought to qualitatively examine the phytochemical components and assess the antibacterial properties of the chloroform extract of M. lucida root on several chosen clinical bacterial pathogens. However, the root extract of M. lucida has not been extensively studied for its antibacterial potential, especially against antibiotic-resistant strains.

Therefore, this study investigates the antibacterial activity of M. lucida root extracts against selected two bacterial strains using in vitro and in silico assays.

2 Methodology

2.1 Materials and Methods

2.1.1 Bacteria

Selected gram-positive and gram-negative bacterial strains include; Staphylococcus aureus and Pseudomonas aeruginosa used in the in vitro antibacterial susceptibility study of M. lucida root crude extracts on the bacterial species. These isolates were subcultured onto sterile nutrient agar plates and incubated for 24 h at 37ºC, and the pure cultures were used for the antibacterial analysis.

2.1.2 Plant Sample

The fresh roots of M. lucida were carefully harvested from the Ebualawe Area, Iwo, Osun State, Nigeria in July 2021. Plant specimens were authenticated and a voucher specimen number was given (BUH036). The roots were washed and air dried for two weeks after cutting into small chunks then homogenized to fine powder which was then stored in airtight containers for further analysis.

2.1.3 Extraction

After macerating 150 g of powdered M. lucida root in 1 L of chloroform for 48 h at room temperature with gentle shaking, the material was filtered through filter paper (Whatman, No. 42). The process was repeated three times to ensure adequate extraction. The extracts were combined and concentrated using a rotary evaporator. Phytochemicals were screened using the procedure outlined by Harborne (1984) [22] before being analyzed, the concentrated extracts were sealed and kept in a refrigerator. The yield of the extract was determined and noted.

2.1.4 Antibacterial assay

The agar diffusion method was used to evaluate the in vitro antibacterial activities of the extracts [23] using a modification of Adeleke and Owoseni [24] protocol and the guidelines of the Clinical Laboratory Standards Institute [25] with slight modifications. 10% DMSO served as the negative control and gentamicin as the positive reference standard medication for the test bacterium. The agar plates were inoculated with the bacteria suspension using a sterile swab on the surface of a Mueller–Hinton agar plate. After applying uniformly, they were allowed to dry for a few minutes. Using sterile forceps, the gentamicin-impregnated disks were placed onto the inoculated agar surface, Precautions was taken to ensure that there was sufficient spacing between the disks and a complete contact between the disk and the agar surface. The inoculated agar plates were incubated at 35 °C for a period of 24 h.

The average zone of susceptibility and average deviation for the test substances against the bacteria were recorded during the duplicate experimentation.

2.1.5 Data organization and statistical analysis

With the aid of Microsoft Word 2016 and Microsoft Excel 2016, the data was arranged and tallied. The studies were performed in triplicate, and the mean and standard deviation (MSD) were determined as the average of the clear zone and standard deviations (SD).

2.2 Molecular docking of Compounds from Plant Extracts of Morinda lucida against selected Microorganisms.

2.2.1 Preparation of compounds

The compounds used for molecular docking studies were retrieved from a previously published work [21]. The Ligprep module of the Schrodinger suit was used to prepare the compounds. The OPLS4 (optimized potentials for liquid simulations) was utilized for force field and energy minimization [26]. The ionization state was established using the Epik ionization tool in ligprep at a pH range of 7.0 ± 1.0 [26]. The 3D conformer structure of the ligands was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/). To prepare the receptor, crystal structures of the different proteins, S. aureus GyrB ATPase, and Pseudomonas aeruginosa DNA gyrase B 24 kDa ATPase subdomain complexed with EBL2888, were retrieved from the protein data bank (PDB) [3U2D and 7PTG] (https://www.rcsb.org).

A grid box was generated around the centroid of the co-crystallized ligand on the prepared protein using the Glide Receptor Grid Generation module of the Schrodinger suit. The grid center coordinates on the receptor were set at 7PTG (x = 18.805600541666667, y = -17.01892358333333, z = -0.7439418333333333) and 3u2d (x = 0.2916116222222224, y = 2.6178757555555547, z = 24.38558124444444) and the inner box defines the diameter midpoint restrain of each ligand docked was set at 12 Å × 12 Å × 12 Å.

2.3 Molecular docking

The prepared compounds were docked using extra precision (XP) glide docking into the produced receptor grid. The force field of choice in the docking computation was the OPLS4 force field.

2.4 Molecular dynamics simulation

The two protein–ligand complexes that showed the best binding affinity for both strains of bacteria studied were selected for molecular dynamics simulation. 2-hydroxy-1-methoxy anthraquinone for S. aureus while alizarin for P. aeruginosa. In the molecular dynamics simulation studies, parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), and Intermolecular hydrogen bonding were taken into consideration.

3 Results and discussion

3.1 In vitro analysis

Figure 1 shows the antibacterial activities of the three extracts tested against two organisms. These were compared with the antibacterial activity of Gentamicin.

Fig. 1
figure 1

Susceptibility of bacteria to M. lucida root extracts

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The extract showed different antibacterial activities against S. aureus and P. aeruginosa. The hexane extract had the weakest activity for S. aureus when compared to other extracts while the other extracts showed excellent susceptibility with chloroform extract having the best susceptibility against S. aureus. Hexane extract has better activity against P. aeruginosa while ethanol extract has a more promising activity against S. aureus.

These results are in agreement with the earlier published reports [2], which confirmed the bioactivities of the aqueous and ethanol extracts of M. lucida against Salmonella typhi, Salmonella paratyphi, Salmonella typhorium, and Escherichia coli. In addition, several parts of M. lucida have proven to possess antibacterial potential. [27]

P. aeruginosa, which is also resistant to different antibiotics [5] had its growth inhibited by ethanol and chloroform extracts of M. lucida.

Burns and wound infections are caused by P. aeruginosa; these infections frequently occur in diabetic patients and those who have recently undergone surgery. Hospital-acquired wound infections are most frequently brought about by S. aureus. [27, 28]

3.2 In silico

The in silico analysis was used to screen and identify the bioactive compounds with the potential to influence the microbial activity noted in the in vitro analysis. The binding affinity, amino residues, interactions, and bond distances for Pseudomonas aeruginosa (7PTG) and Staphylococcus aureus (5TW8).

3.2.1 Pseudomonas aeruginosa (7PTG)

It is evident from the interaction of the quinones with the 7PTG protein as seen in Table 1 that gentamicin (Fig. 2) had the highest binding affinity with a docking score of − 7.79 kcal/Mol which indicates that it has the best binding to the receptor. Gentamicin forms multiple hydrogen bonds with the following amino acid residues: Glu44, Asp51, Asn48, Ile98, Val99, Leu100, Ile96, and Gly121. It also shows electrostatic interactions with negatively charged Asp residues, forming salt bridges (Fig. 2). The variety of hydrogen bonds and electrostatic attractions with key binding site residues lead to its tight binding. [28]

Table 1 Binding affinity, amino residues, interactions, and bond distances for Pseudomonas aeruginosa (7PTG)
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Fig. 2
figure 2

Gentamicin interacting with amino acid residues of 7ptg protein

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From the quinones identified in M. lucida root extracts, all the compounds with the hydroxyl and methoxy functional groups had bonding affinity ranging from − 5.65 to – 3–85 kcal/mol (Figs. 3, 4). 1-Hydroxy-4-methylanthraquinone (− 5.47 kcal/mol) (Fig. 5) and 1-Hydroxy-2-methylanthraquinone (− 5.59 kcal/mol) (Fig. 6) show affinity with amino acid residues such as Thr167 and other hydrophobic interactions. The interaction of the methyl derivatives 9,10-Anthracenedione, 2-methyl- (Fig. 3) and 9,10-Anthracenedione, 1-methyl- (Fig. 4) interacting with amino acid residues indicates that the presence of the methyl group did not effectively increase the interaction as the hydroxyl groups with amino acid residues of the 7PTG protein.

Fig. 3.
figure 3

9,10-anthracenedione, 2-methyl-interacting with amino acid residues of 7ptg protein

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Fig. 4.
figure 4

9,10-anthracenedione, 1-methyl-interacting with amino acid residues of 7ptg protein

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Fig. 5.
figure 5

1-hydroxy-4-methylanthraquinone interacting with amino acid residues of 7ptg protein

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Fig. 6.
figure 6

1-hydroxyl-2-methylanthraquinone interacting with amino acid residues of 7ptg protein

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Others such as 3-Hydroxyl-1-methoxyanthraquinone (Fig. 9) had an affinity of -3.85 kcal/mol, 2-hydroxyl-1-methoxyanthraquinone (Fig. 7) − 5.65 kcal/mol, and 9,10-Anthracenedione, 1,5-dimethoxy- (Fig. 8) − 4.54 kcal/mol.

Fig. 7.
figure 7

2-hydroxyl-1-methoxyanthraquinone interacting with amino acid residues of 7ptg protein

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Fig. 8.
figure 8

9,10-Anthracenedione, 1,5-dimethoxy-interacting with amino acid residues of 7ptg protein

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The methoxy substituted anthraquinones like 1,5-dimethoxy anthraquinone (− 4.54 kcal/mol) and 1,8-dimethoxy anthraquinone (− 5.56 kcal/mol) (Figs. 9, 10 and 11 respectively) had an affinity with similar amino acids except for VAL 73. The methyl-substituted anthraquinones have varied affinities which that the position of the methyl group influences that binding affinity.

Fig. 9.
figure 9

3-hydroxyl-1-methoxyanthraquinone interacting with amino acid residues of 7ptg protein

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Fig. 10.
figure 10

1,8-dimethoxyanthraquinone interacting with amino acid residues of 7ptg protein

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Fig. 11
figure 11

Alizarin interacting with amino acid residues of 7ptg protein

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Naturally occurring alizarin (Fig. 11) shows the highest binding energy among the natural anthraquinone compounds from M. lucida (− 6.2 kcal/mol) owing to multiple hydrogen bonds, consistent with its antibiotic potential as described by Khan et al. [29]. Thus it can be predicted that alizarin contributes strongly to the susceptibility properties of M. lucida against Pseudomonas aeruginosa.

3.2.2 Staphylococcus aureus (5TW8)

Table 2 presents the results of the docking for S. aureus showing the affinities, distance, and natures of bonds. Gentamicin (Fig. 12) shows the highest binding affinity of − 7.04 kcal/mol, consistent with its potent antibacterial activity against S. aureus. [30]

Table 2 Binding Affinity, Amino residues, interactions, and bond distances for Staphylococcus aureus (5TW8)
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Fig. 12
figure 12

Gentamicin interacting with amino acid residues of 5tw8 protein

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2-Methyl (Fig. 13) and 1-methyl (Fig. 14) substituted anthraquinones have weaker affinities than Gentamicin with − 3.07 and − 3.67 kcal/mol respectively due to limited hydrophobic contacts.

Fig. 13.
figure 13

9,10-anthracenedione, 2-methyl-interacting with amino acid residues of 5tw8 protein

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Fig. 14.
figure 14

9,10-anthracenedione, 1-methyl-interacting with amino acid residues of 5tw8 protein

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Alizarin (Fig. 21), 1-Hydroxy-4-methylanthraquinone (Fig. 15), and 1-hydroxy-2-methylanthraquinone (Fig. 16) showed improved binding affinities (− 3.7, − 3.8, and − 4.36 kcal/mol respectively) over the methyl anthraquinones, attributed to additional hydrogen bonds with binding site residues like Tyr291. The hydroxy group enhances their antibacterial potential as noted experimentally. [31]

Fig. 15.
figure 15

1-hydroxy-4-methylanthraquinone interacting with amino acid residues of 5tw8 protein

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Fig. 16.
figure 16

1-hydroxyl-2-methylanthraquinone interacting with amino acid residues of 5tw8 protein

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9,10-Anthracenedione-2-hydroxy-1-methoxy (Fig. 17) also shows a higher binding energy (− 5.5 kcal/mol) than the other hydroxyl-methyl derivative of anthraquinone, facilitated by hydrogen bonding interactions. The dimethoxy anthraquinones 1,5-dimethoxyanthraquinone (Fig. 18), 3-Hydroxyl-1-methoxyanthraquinone (Fig. 19), and 1,8-dimethoxyanthraquinone (Fig. 20) have lower affinities than the other anthraquinone derivatives potentially due to steric effects.

Fig. 17.
figure 17

2-hydroxyl-1-methoxyanthraquinone interacting with amino acid residues of 5tw8 protein

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Fig. 18.
figure 18

9,10-Anthracenedione, 1,5-dimethoxy-interacting with amino acid residues of 5tw8 protein

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Fig. 19.
figure 19

3-hydroxyl-1-methoxyanthraquinone interacting with amino acid residues of 5tw8 protein

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Fig. 20.
figure 20

1,8-dimethoxyanthraquinone interacting with amino acid residues of 5tw8 protein

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Naturally occurring 2-hydroxy-1-methoxyanthraquinone (Fig. 17) shows the highest binding energy among the natural anthraquinone compounds from M. lucida (− 5.5 kcal/mol) owing to multiple hydrogen bonds with amino acids such as ASN 141, ALA 74, LEU 115, SER 116.

2-hydroxyl-1-methoxyanthraquinone isolated from the stem bark of M. lucida has been reported to possess antibacterial activities. [32]. Figure 21, shows the binding interractions of alizarin with amino acids in the 5tw8 protein. Alizarin (Fig. 21)showed improved binding affinities (− 3.7 kcal/mol ) attributed to additional hydrogen bonds.

Fig. 21
figure 21

Alizarin interacting with amino acid residues of of 5tw8 protein

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3.3 Molecular dynamics (MD) simulations

3.3.1 The root mean square deviation (RMSD) of protein–ligand

RMSD of S. aureus (5TW8) in complex with 2-hydroxy-1-methoxy anthraquinone and P. aeruginosa (7tgp) in complex with alizarin is shown in Figs. 22 and 23. The RMSD of protein–ligand was done for 50 ns. The RMSD value of 2-hydroxy-1-methoxy anthraquinone with 5TW8 is stabilized at 1.5 Å and 20 ns and remains stabilized for 50 ns (Fig. 22). Similarly, in the case of alizarin complex with 7tgp is stabilized at 5 Å and 5 ns and remains unstable for 50 ns (Fig. 23). The RMSD of the ligand with respect to the alpha carbon (prot_CA) of the protein with the reference frame shows that in Fig. 22 the protein is relatively stable and is not undergoing major conformational changes which could disrupt activities such as catalysis. It is expected that a protein being in the range of 3 Å as expected for non-globular proteins [33]. The RMSD explains how the ligand interacts with the receptor and the stability over time [34]. The alizarin ligand in the binding site of the 7tgp protein is not very stable as seen in Fig. 23.

Fig. 22
figure 22

RMSD graph of S. aureus (5TW8) in complex with 2-hydroxy-1-methoxy anthraquinone for 50 ns

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Fig. 23
figure 23

RMSD graph of P. aeruginosa (7PTG) in complex with alizarin for 50 ns

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3.3.2 The root mean square fluctuation (RMSF) of protein–ligand

To determine the flexibility of protein structures and determine the deviation of the particles from the original position of macromolecules (3-D structure of the protein) the RMSF is used [35]. As seen in Fig. 24, low fluctuations are observed for 5TW8 in a complex with 2-hydroxy-1-methoxy anthraquinone between 0.5 and 2.0 Å. Similarly, 7tgp in complex with alizarin also showed low fluctuation between 0.5 and 2.5 Å (Fig. 25). Thus, the above results revealed that RMSF plots for the two complexes and ligand RMSF have stability within the binding pocket of the target RMSF. [35]

Fig. 24
figure 24

RMSF graph of 5TW8 in complex with 2-hydroxy-1-methoxy anthraquinone for 50 ns

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Fig. 25
figure 25

RMSF graph of P. aeruginosa (7PTG) in complex with alizarin for 50 ns

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Figures 26 and 27 further confirm the stability of the proteins by showing interaction fractions of the various residues of the protein by which the amino acids bond to the various ligands. SER 116 had the highest interaction fraction for 5TW8. SER had 87% interaction with the hydroxyl group of 2-hydroxy-1-methoxy anthraquinone (the ligand) while the oxygen on the methoxy group of the ligand had a 60% interaction (Fig. 28). ASP 75 shows the highest interaction fraction for 7PTG with water bridges. The most common type of interaction between alizarin ligand and the 7PTG protein is hydrophobic interactions. Figures 28 and 29 buttresses the nature of the most important bonding. The presence of intramolecular hydrogen bonding as seen in Fig. 29.

Fig. 26
figure 26

Contact Histogram of 5TW8 showing the various interaction fractions of the amino acids

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Fig. 27
figure 27

Contact Histogram of 7PTG showing the various interaction fractions of the amino acids

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Fig. 28
figure 28

Contact summary for 5TW8

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Fig. 29
figure 29

Contact summary for 7PTG

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4 Conclusion

Since these bacteria are of public health concern and can produce toxins that are stable under heat [36], utmost care must be taken to properly address them. One of which is the identification of compounds from plants with antibacterial properties. As seen in the in vitro results, the extracts of M. lucida had good susceptibility properties against the two drug-resistant bacteria when compared to gentamicin. From the anthraquinones identified in M. lucida, 2-hydroxy-1-methoxy anthraquinone is predicted to have a better susceptibility with S. aureus while alizarin has better susceptibility properties against P. aeruginosa. The MD shows a notable level of stability between the protein and the ligands in this study.

Thus anthraquinone compounds from M. lucida show that they have bioactive potentials as antibacterial drugs. This provides a justifiable reason for further identification and characterization of all the compounds that can serve as enzyme inhibitors with potential medicinal applications. Therefore future research would be on the isolation and characterization of secondary metabolites which have been identified in the study to be responsible for the antibacterial properties .