Explication of Pharmacological Proficiency of Phytoconstituents from Adansonia digitata Bark: An In Vitro and In Silico Approaches

Compared to other drug discovery sources, traditional medicine has significantly contributed to developing innovative therapeutic molecules for preventive and curative medicine. The Baobab tree, also known as Adansonia digitata L., is significant in Africa due to its multitude of benefits and various parts that serve different purposes, providing economic support to rural communities. The analysis of a plant sample using Fourier transform infrared (FT-IR) spectroscopy detected multiple functional groups, such as carboxyl and aromatic groups. Additionally, gas chromatography-mass spectroscopy (GC-MS) was utilized to identify various compounds present in the sample, including tetrachloroethylene and octyl ester. The results of different assays, such as α-diphenyl-β-picrylhydrazyl (DPPH), superoxide, nitric oxide scavenging assays, and total antioxidant by thiobarbituric acid method (TBA) and ferric thiocyanate (FTC) method, demonstrated a substantial scavenging of free radicals and an effective antioxidant efficacy. The bark's antimicrobial activity was tested through agar diffusion, resulting in a range of zone of inhibition from 10.1 ± 0.36 mm to 20.85 ± 0.76 mm. The minimum inhibitory concentration (MIC) value was observed to be approximately 0.625 µg/mL. The biofilm inhibition percentage ranged from 9.89% to 57.92%, with the highest percentage being 57.92%. The GC-MS and FT-IR studies revealed phytocompounds, which were then analyzed for their potential therapeutic properties. Computational studies were conducted on the phytocompounds against Pseudomonas aeruginosa and C2 kinase (antioxidant). The study concluded that the Adansonia digitata bark extract and its phytocompound have potential therapeutic efficacy against the target proteins. The best docking scores were about −7.053 kcal/mol and −7.573 kcal/mol for Pseudomonas aeruginosa and C2 kinase (antioxidant), respectively. The interaction patterns with the crucial amino acid residues elucidate the inhibitory efficacy of the phytocompounds.


Introduction
Medicinal herbs have been a valuable natural resource for life-threatening remedial diseases since antiquity [1].Tese medicinal plants are regarded as good sources of ingredients for drug development and synthesis.Diferent medicinal plants can treat similar illnesses based on geographical occurrence [2].Ethnobotanical research is required to statistically document the use of medicinal plants and their therapeutic and toxicological consequences.With 80% of all synthetic medicines produced, it is still signifcant today as a source for drug discovery and the primary healthcare technique for around 85% of the world's population [3].Te baobab tree, Adansonia digitata L., is a native of Africa.In most tropical Africa, A. digitata (African baobab) is one of the most signifcant wild food plants and a vital species with considerable ecological and social value.It is one of nine global baobab species in the Adansonia genus.Diferent solvent extracts have been shown to have therapeutic characteristics [4].Studies have shown that extracts derived from the Adansonia digitata tree's bark efectively treat infammation, diabetes mellitus, and microbial infections.Te bark of this tree contains a variety of favonoids, some of which are marketed in Europe under the name "cortex cael cedra" and used as a treatment for fever, and a substitute for cinchona bark [5].
Te tree's bark contains several phytoconstituents with potential medicinal properties, including tannins, saponins, favonoids, alkaloids, phytosterols, glycosides, and terpenoids [6].Flavonoids are a type of polyphenolic compound that contains favones [7].Tey are composed of aromatic rings linked to three carbon atoms and cyclic structures with oxygen.Tey are currently used in traditional medicine to treat malaria, gastrointestinal ailments, and other diseases and infections [8].It is a large, versatile fruit-bearing tree with over 300 traditional applications and commercial values [9].Te fruit pulp has a large amount of vitamin C, proteins, calcium, phosphate, potassium, carbs, fbers, and lipids and can be used as an appetizer or drink.Seeds are high in lysine, thiamine, calcium, and iron, including signifcant sodium, phosphorus, zinc, iron, magnesium, and manganese [10].In the southern part of India (specifcally Tamil Nadu), Adansonia digitata dotting the Ramanathapuram shoreline (Map: 13.18325184557891, 80.30669745948643) stands out with massive trunks and lush foliage.Baobabs cannot thrive in wet locations and cannot withstand heavy rain.Tey are only found in southern Tamil Nadu (Chennai, Madurai, and Ramanathapuram), where the climate is suitable for survival.Adansonia digitata shows potential as a useful source for drug development and primary healthcare approaches for an extensive percentage of the world's population when implementing consideration of traditional applications and contemporary scientifc investigations on the therapeutic characteristics of the plant.However, further investigation and ethnobotanical recording need to be undertaken to grasp and exploit its promise for life-threatening restorative disorders completely.
Te main aim of this study is to investigate the physicochemical characteristics and chemical composition of the bark extracts, which can be achieved using analytical tools, namely, phytochemical screening, gas chromatographymass spectroscopy (GC-MS), and Fourier transform infrared (FT-IR) studies.Tis investigation's primary objective is to thoroughly examine the biological and antibacterial efectiveness of an extract obtained from A. digitata, also referred to as the baobab tree.We propose an integrated strategy that combines in vitro and in silico techniques to accomplish the proposed study.Our objective is to evaluate the Adansonia digitata ethanol extract (ADEE) efcacy against several diseases, including pathogenic microbes that form bioflms. Te study seeks to ascertain the extract's efectiveness in inhibiting the bioflm formation of Pseudomonas aeruginosa by observing the development and morphology of bioflms under these circumstances.We aimed to obtain an in-depth comprehension of the extract's potential in diferent biomedical applications by carrying out precise in vitro and using advanced computer simulations, including molecular docking and dynamics simulation, ultimately incorporating our understanding of its therapeutic and pharmaceutical prospects.

Stem Bark Collection and Extraction. Te stem bark of
Adansonia digitata was collected from Tangachimadam, Ramanathapuram District, Tamil Nadu, India, using a sterile knife.Before being crushed into powder with a sterile pestle and mortar in a laboratory, the bark was thoroughly washed with distilled water and then air-dried in the shade for two weeks [11].To extract secondary metabolites from the A. digitata bark, ethanol was utilized.Te extraction process involved soaking 100 grams of powdered bark in 1000 mL of ethanol for two days at room temperature with intermittent shaking.Te resulting mixture was then fltered, and the fltrate was concentrated using a rotary evaporator until a solid residue was formed.Te concentrated sediment was stored at a refrigerated temperature of 4 °C until required [12].

Assessment of Antioxidant Activity.
Endogenous enzymatic antioxidants, such as superoxide dismutase (SOD), catalase (CAT), and phospholipid hydroperoxide glutathione peroxidase (GPx), serve as the frst line of defense against free radicals, while nonenzymatic antioxidants, such as glutathione, vitamin C, and vitamin E, act as the second line of defense [13].Te antioxidant activity of ADEE was assessed in vitro through several methods, including scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, nitric oxide scavenging, and measurement of superoxide anion scavenging activity.Tese tests were conducted following the methodology outlined by Kannaian et al. [14].Additionally, the protective role of the ethanol extract in inhibiting free radical-mediated DNA-sugar damage was evaluated by estimating the total antioxidant activity of ADEE using ferric thiocyanate (FTC) and thiobarbituric acid (TBA).Vitamin C (ascorbic acid) was used as the control for this study.A brief explanation of the methodology was included in Supplementary File 1.  [15].After incubation, we were able to assess the remarkable antimicrobial activity of ADEE.Te presence of a clear zone around the well proved its efectiveness in inhibiting microbial growth.Tese data highlight the potential of ADEE has potential to combat harmful pathogens.Te observed zone of inhibition was measured in mm.

Minimum Inhibitory Concentration (MIC).
Te lowest concentration of antimicrobial factor inhibits the growth of microbes after the incubation of 24 hours, referred to as minimum inhibitory concentration (MIC), and the lowest concentration required to inhibit microbial growth [16].MIC was performed on the ADEE against bacterial isolates such as Pseudomonas aeruginosa (positive control and negative control) by using tetracycline as standard.Te extract solution from the frst test tube and 0.5 ml of nutrient broth medium (NB) were added to each tube, which was then homogenized (including sample preparation concentration) [17].Each well was flled with dissolved 2,3,5-triphenyltetrazolium chloride (TTC), which was then incubated at 37 °C for 30 min in the dark to detect respiratory activity (bacterial growth).TTC is a salt that may transform from clear to pink when digested by living bacteria.Te MIC of the organism was determined to be the lowest concentration that displayed turbidity.[22].Additionally, Z-score was calculated to analyze the overall structural quality of the modeled structure, which was executed in the Protein Structure Analysis (ProSA) server [23].By using the template structure from Protein Data Bank (PDB ID-5OE3), the structure was modeled and superimposed.Te biological targets of P. aeruginosa and C2 kinase (antioxidant) were exposed to decipher the inhibitory efects of the phytocompounds retrieved from GC-MS analysis.Te Protein Preparation Wizard is used to prepare the target structure by adding missing atoms, correcting bond orders, and adding hydrogen atoms based on protonation states.Additionally, water molecules were removed beyond 5 Å followed by adding the missing side chain [24].Te Protein Preparation Wizard automates these processes to assist in making sure that the protein structure is in an acceptable state for the next investigations, including molecular docking, molecular dynamics simulations, and structure-based drug discovery.

Lead Optimization.
Te 2D structure of the chosen phytocompounds was retrieved from the PubChem database in Structured Data File (SDF) format.Te LigPrep module assists in generating the possible tautomers and stereochemical forms of the compounds by using the Optimized Potentials for Liquid Simulations (OPLS_2005) force feld, which helps to minimize the steric clashes of phytocompounds followed by adjusting bond angles, lengths, and torsional angles.Epik calculates protonation and tautomerization states based on a database of pKa values and rules at a pH of 7.0 ± 2.0 [25].

Molecular Docking.
Molecular docking is an extensive platform used to envisage the binding interaction between the target and the small molecule, which predicts the conformation and orientation of the ligand binding at the protein's active site [26].Te most favorable binding mode has the lowest docking score with higher precision at the binding site of the target while the docked ligand with the best score is often considered as the lead molecule for further validation.Docking was executed in extra-precision (XP) mode on the Glide module in Schrodinger [27].Further docking was executed via the PDB entry with a cobound ligand that possesses the highest ligand similarity score to the query ligand to validate the docking process.
Partial atomic charges were considered while all other parameters were running in the default manner by adding hydrogen atoms at the pH of 7.0.Grid generation is a key phase in molecular docking studies because it determines the region in which the ligands will be investigated, and signifcantly improves the precision and dependability of the docking outcomes.Te grid was generated over the active site residues of the target proteins with X, Y, and Z coordinates [28].By carefully assessing the docking score and thoroughly analyzing the interaction patterns that indicate a signifcantly stronger binding afnity, we assuredly selected specifc leads for further analysis.

Molecular Mechanics with Generalized Born and
Surface Area (MM/GBSA).Te energies of the free ligand and receptor are altered by their contact (GBind), and these energies also have a signifcant impact on the stability of the complex.Molecular mechanics with generalized born and surface area (MM/GBSA) forecasts the rate at which the interaction can be between the chosen ligand and protein, which is determined by kcal/mol [29].MM-GBSA was performed on the Prime module in Schrodinger, which was driven by the OPLS_2005 force feld in the variable solvent generalized born (VSGB) solvation model.Te following equation was employed for energy calculation in MM-GBSA: where ∆EMM represents the minimized energy of the docked complex, ∆GSolv represents GBSA and the sum of the solvation energy of the complex, and ∆GSA represents the surface area energy of the complex.

Toxicity Prediction.
Our dependence on these chemicals has reached critical levels, and failure to take decisive action now could have calamitous consequences for our health and environment.Bioavailability is a crucial factor for any oral drug candidate [30].A high bioavailability score improves the binding selectivity profle and reduces undesirable drug efects.According to the duration and severity of the chemical exposure, this sort of exposure can be both adverse and benefcial.Terefore, it is crucial to experimentally confrm the compounds' and their combination's potential for toxicity.Te selected phytocompounds were subjected to toxicity studies using ProTox-II Server [31].Te classifcation of the prediction tactics into various levels of toxicity, such as oral toxicity, organ toxicity (hepatotoxicity), toxicological endpoints (mutagenicity, carcinogenicity, cytotoxicity, and immunotoxicity), toxicological pathways (AOPs), and toxicology targets, ofers an understanding of the possible molecular mechanisms underlying such hazardous reactions.

Molecular Dynamics Simulation (MDS).
Molecular dynamics simulation (MDS) is a wide method used for simulating and evaluating the physical motions of atoms and molecules [32].When the atoms and molecules are allowed to interact for a certain period, the system's dynamic progress can be observed.MDS was performed on the GROningen MAchine for Chemical Simulations (GROMACS) package for both apo and complex forms.Te system was subjected to a 100 ns period with the cubic box followed by solvating the system by adding a signifcant amount of Na and Cl ions [33].
Te whole system was minimized for structural relaxation at 50,000 steps using the Steepest descent algorithm, besides the equilibration of solvent and ions throughout the complex and apo forms under a constant number of particles, volume, and temperature (NVT) by using a method of Berendsen thermostat coupling with the coupling set at 0.1 ps at 300 K. Additionally, the constant number of particles, pressure, and temperature (NPT) ensemble was utilized to equilibrate the system using the Parrinello-Rahman pressure coupling method with the coupling constant at 2.0 ps at a pressure of 1.0 bar [34].Finally, the fnal production of MD was performed for apo form at 50 ns and the generated trajectories were evaluated for further analysis to predict the stability of the modeled structure.Future studies will be focused on assessing the stability of the docked complex through MDS.

In Vitro Antioxidant
Studies.In the human system, free radicals are produced, and they exist in various forms, including hydroperoxyl, peroxyl, superoxide, hydroxyl, and alkoxyl radicals.Tese free radicals were removed with the help of natural antioxidant enzymes.Antioxidants are the primary defense mechanism of the human body and perform lipid peroxidation inhibition.Tey scavenge free radicals, stopover toxic oxidation product fabrication, and uphold nutritional quality.Te overall fndings of antioxidant studies of Adansonia digitata ethanol extract (ADEE) are shown in Figure 1 and Table 1.

2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Free Radical
Trapping Activity.ADEE free radical trapping activity through the DPPH method was performed at various concentrations from 50, 100, 150, 200, and 250 µg/mL.Results signify that ADEE traps more than 50% of free radicals, proving that the extract has tremendous free radical trapping activity.Te results were well correlated with the standard ascorbic acid.

Nitric Oxide Trapping Activity. ADEE was represented
by nitric oxide radical strapping activity in various concentrations (15,30,45,60, and 75 µg/mL).Tese assay results displayed the signifcant inhibition of free radicals, comparable to the standard ascorbic acid.Te outcome of this parameter suggests that the extract might be an efective peroxide inhibitor.

Superoxide Anion-Trapping Activity.
Te inhibition of ADEE was achieved in a dose-dependent manner by superoxide radicals, and the resulting consequences were observed.Te outcomes exhibited that the extract pointedly repressed the superoxide radicals at concentrations of 15, 30, 45, 60, and 75 µg/mL.In the 75 µg/mL concentration, more than 50% inhibition of superoxide was observed, and this remarkable inhibition was comparatively well with the standard ascorbic acid.

Total Antioxidant Activity.
Te total antioxidant activity can be measured by two methods, FTC and TBA.Te FTC method can measure the production of peroxide during the early phase of oxidation, while the TBA method can be used to measure the secondary products of oxidation, such as aldehyde and ketone.

Antimicrobial Activity.
Te dose levels of 25, 50, 75, and 100 µg/mL were employed to estimate the antimicrobial activity of ADEE.Te antimicrobial activity was performed against Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas aeruginosa, and Staphylococcus aureus with the dosage of 25, 50, 75, and 100 µg/mL.Te zone of inhibition against bacterial pathogens through the agar well difusion method reveals that ADEE has remarkable antibacterial efcacy, and the results are shown in Table 2 and Figure 2. Te results showed that P. aeruginosa showed the highest zone of inhibition (20.85 ± 0.76) at 100 µg/mL concentration compared with the other isolates.ADEE inhibits the growth of selected microbes with better inhibition values.Te antibacterial properties of ADEE could be due to phytochemicals, such as alkaloids, tannins, saponins, favonoids, and terpenoids, which are present in A. digitata bark.Te diferences between the groups were analyzed using one-way ANOVA, followed by Duncan's test for multiple comparisons (SPSS 16.0).Statistically signifcant values are represented in Table 2.

Minimum Inhibitory Concentration (MIC).
Te minimum inhibitory concentration (MIC) is a crucial measure that determines the lowest concentration of a drug needed to effectively inhibit the growth of microorganisms.Tis is accurately measured by observing turbidity in a test tube, and understanding the MIC is essential in determining the drug's efectiveness.According to the well difusion method results, ADEE may be very efective at inhibiting the growth of P. aeruginosa.By using twofold serial dilution techniques, the MIC was established.Te MIC values and percentage yield of the ADEE against P. aeruginosa are shown in Supplementary Figure 1 and Table 3. Trimethyl tetrazolium chloride (TTC) is used in this technique to indicate viable microbes.Tubes 4 and 5 have the yellowish color of the broth, whereas tubes 6 and 7 have a slight reddish shade as a gradual shift into a cherry red color.Tube 5 is the minimal inhibitory action with a 0.625 µg/ mL concentration range.Tis endpoint was considered for the bioflm-inhibiting concentration range for P. aeruginosa, which was signifcantly correlated with the standard.

Pseudomonas aeruginosa
Figure 2: Antimicrobial activity of Adansonia digitata ethanol extract (ADEE) against bacterial pathogens using diferent concentrations.Scientifca P. aeruginosa.After diluting the sample, diferent concentrations ranging from 1.56-0.003µg/mL were added to 10 wells with positive and blank controls.Crystal violet was used to visualize the bioflm information.Well 1, with the highest concentration of extract, had the highest percentage of bioflm inhibition (57.92%), and well 10, with the lowest concentration of ADEE, had the lowest percentage of bioflm inhibition (9.89%).Te absorbance of both planktonic cells and crystal violet staining was measured at 630 nm and 492 nm.Te observed results were signifcantly correlated with the standard (tetracycline) values.

Assessment of Pellicle Formation. PAO1 gene in
Pseudomonas aeruginosa coded for pellicle production (a thin bioflm around cells that forms at the air-liquid interface of a standing liquid culture).Inhibiting the formation of pellicles is necessary because it can aid in preventing the development of bioflms, which are surface-associated microbial populations enclosed in an extracellular matrix in P. aeruginosa, which could diminish severe infections.Te treatment of ADEE (35 µg/mL) notably reduced pellicle formation at 12 µg/mL markedly condensed pellicle development was experimental in PAO1 (Figure 3).

Fourier Transform Infrared Spectroscopy. Te FT-IR analysis revealed ADEE's peak values and functional groups'
presence in the bark ethanolic extract.Te various functional groups observed in the ADEE indicate the presence of ketone, carboxylic acid, silicon, phosphate, alkanes, chloro, bromo, and hydroxy compounds, and the plot is illustrated in Supplementary Table 1 and Figure 4. Te presence of functional constituents in the given extract was analyzed fawlessly using the FT-IR spectrum, which may support the extract's biomedical properties and therapeutic efcacy.

GC-MS. GC-MS results
of ADEE exhibited that different phytocompounds were identifed.Te report showed fve major peaks (5-octadecene; (E)-pentanoic acid, 5-hydroxy-, 2,4-di-t-butyl vinyl esters; 1-nonadecene; 5eicosene; 1-octadecanol) from the ethanol extracts.Te best hits were selected based on their biological properties, therapeutic efcacy, and peak range.ADEE showed better phytocompounds, and these extracts were subjected to further investigations.Results and fndings from ADEE are shown in Supplementary Table 2, and the chromatogram is displayed in Supplementary Figure 3.

Target Validation.
Te pseudomonas quinolone signal (PqsA) structure was modeled using the template while the superimposed structure was visualized using PyMOL.Furthermore, the model was validated using the Ramachandran plot to gain insights into the modeled structure.Te regions signifcantly occupied are the alpha-helices and beta-sheets, which are typical secondary structures in proteins.Te Ramachandran plot depicted that 95.2% region is occupied by the most favored region while the disallowed region was found to be 0.0%, which is shown in Figure 5. Additionally, the Z-score value was found to be −8.79 and the modeled structure was superimposed with the template, resulting in an RMSD value of approximately 0.23 Å.
3.9.Apo Dynamics of the Modeled Structure.Apo dynamics aid in understanding the role of protein performance in its natural environment, regulation, and interaction with other molecules.Te development of more potent therapeutics is rendered feasible by the utilization of dynamic regions in the apo form that regulates the generation of small molecules or drugs that target certain protein conformations or states.Te modeled structure of P. aeruginosa was subjected to molecular dynamics simulation by utilizing the GROMACS package employing the GROMOS96 43a1 force feld.Figure 6 illustrates the root-mean-square deviation (RMSD) and root-mean-square fuctuation (RMSF) plot, which deciphers the overall structural stability of the modeled structure.Te average RMSD value was observed at 0.25−0.3nm, which was under the threshold, and initially, mild deviation was observed at 0.25 nm and reached the 8 Scientifca maximum at 0.3 nm.Interestingly, the RMSD plot interprets the stability throughout the simulation period without any aggressive deviation while the average fuctuation rate was observed at 0.15-0.25 nm.Te maximum fuctuation rate was detected on 100-125 residue at 0.5 nm, which occurred at the loop region and did not cause any structural changes or afect the structural stability of the complex.Te overall MDS fndings concluded that the modeled structure was stable without much deviation and fuctuation.Hence, it was considered for further analysis.

Interaction between Pseudomonas aeruginosa Phytocompounds. Identifying novel targets for
Pseudomonas aeruginosa infection management may be possible by better fguring out the quorum sensing processes that restrict bioflm formation.From the in vitro experiments, P. aeruginosa showed the highest inhibitory activity when compared with the other isolates, while the bioflm formation was inhibited by the bark extract.Te chosen phytocompounds were docked with the modeled structure of P. aeruginosa (pqsA) using the template PDB ID-5OE3.Out of selected phytocompounds, fve docked complexes (559495, 22217550, 550931, 595387, and 6423866), which are illustrated in Figure 7 and tabulated in Table 5, showed a better docking score of about −7.053, 6.743, −5.745, −5.149, and −4.963 kcal/mol, respectively, while the interacting residues formed hydrogen bond, salt bridge, and Pi-Pi interaction with the key residues.Tree hydrogen bond   Te potential for the phytocompounds to be efective as potent ligands for the target protein has been demonstrated by docking investigations that indicate signifcant scores and interactions with key amino acid residues.Tis insight can assist in researching and developing novel drugs since it identifes the phytocompounds that are most probable to bind to the target and perhaps modify its activity.   .By exploring the interactions of 2OXX_559495, it was noted that the hydroxyl group and amine group made interaction with Hie_160 (2.21 Å) and Asp_175 (1.66 Å) and formed hydrogen bond interaction, while salt bridge interaction was noted at Asp_175 (1.66 Å) to interact with the amine group.Second, the 2OXX_22217550 complex exhibited two hydrogen bond interactions with Tyr_115 (2.62 Å) and Val_45 (2.16 Å), which were found to be in contact with the hydroxyl and amine groups.

Interaction of 2OXX_Phytocompounds
Te complex 2OXX_106994 deciphers the hydrogen bond formation with Asp_175 and Lys_68 at the distance of 1.85 Å and 1.95 Å, while Lys_68 (4.53 Å) also interacts with the oxygen group by forming salt bridge interaction, which favors the stability, conformational rigidity, and folding of the target structure.Te other two complexes 550931 and 20393 interacted through hydrogen bonds with Lys_68 (2.05 Å and 2.60 Å) and Asp_175 (2.51 Å), and those interactions were found to be made with the oxygen group.Te overall docking results depicted in Figure 8 and Table 6 concluded that the resultant phytocompounds showed good docking scores interacting with the crucial amino acid residues within the active site of the target.Te highest docking score suggests a strong or favorable binding afnity subsidizing protein-ligand stability.Hence, the study concluded that the phytocompounds docked with the modeled structure of P. aeruginosa and 2OXX were found to be potential or efective ligands against the target.Both in vitro and in silico analyses suggest that the phytocompounds in A. digitata extract exhibited their potent activity as drug candidates and pharmacological efcacies.

MM-GBSA.
Te docked and binding efcacy of the complex was further validated by binding free energy calculation by implementing MM-GBSA.Table 7 demonstrates the calculated energy values of the docked complex of the modeled structure and 2OXX_Complex.Te molecule that needed the least energy to ft comfortably inside the protein's active region is considered to be the best for inhibition or activation; additionally, the stability of the docked complex is signifcantly infuenced by MM-GBSA.Overall, negative energies signify a higher level of stability.Te calculated MM-GBSA scores of the modeled structure with the phytocompounds were identifed as 559495 (−47.40 kcal/mol), 22217550 (−43.91 kcal/mol), 595387 (−39.87 kcal/mol), 6423866 (−50.42 kcal/mol), and 550931 (−40.55 kcal/mol), respectively.
Followed by 2OXX complexes showed better binding free energy, which were well interrelated with the docking scores.Te interactions such as hydrogen bonds, salt bridges, and Pi interactions favored the stability of the docked complexes, whereas the MGBSA scores decipher the precise assessment of bonding afnities of the complex.Te calculated MM-GBSA values of 2OXX with the phytocompounds have the average value observed between −40.67 and −50 kcal/mol, respectively.In conclusion, the MM/ GBSA estimation defned the binding afnities of the highest and lowest compounds in the precise positions as docking energy although there were minor modifcations in the order for compounds holding intermediate docking energy.Future studies will be enabled to further validate the compound stability by MDS, and we have taken all of the chosen complexes.
3.12.Toxicity Prediction.We determined the toxicity parameters of the phytocompounds using ProTox-II Server (Table 8).Te GHS guidelines classify substances into six groups, with the highest group having the lowest likelihood of being dangerous or damaging.Classes 1 (LD50 ≤ 5) and 2 (5 < LD50 ≤ 50) indicate that they are lethal and very certainly will result in death.In this analysis, it was determined that none of the chemicals belonged to classes 1 or 2. Class 3 indicates potential toxicity hazards from oral exposure (50 < LD50 ≤ 300), and compound_595387 belongs to Class 3. Class 4 (300 < LD50 ≤ 2000) classifes compounds that are most likely to be dangerous when consumed; in this case, 22217550, 559476, 6423866, 550931, 559495, and 20393 were found to be in Class 4. Compound_106994 falls in Class 6 (LD50 > 5000), which determines the nontoxic nature of the phytocompound.Te toxicity of phytocompounds was assessed using toxicity criteria like mutagenicity, carcinogenicity, hepatotoxicity, immunogenicity, etc., which were all found to be inactive in most of the phytocompounds.

Discussion
Plant-derived bioactive substances known as phytochemicals have been associated with reducing chronic degenerative diseases in humans [35].Nigella sativa extract contains thymoquinone, which can efectively upregulate and downregulate several biomarker signaling pathways, thereby providing a potential therapeutic target for TNBC [36].Numerous studies have documented the potential of isoliquiritigenin, derived from Glycyrrhiza glabra, to impede the expression of the NF-κB pathway.Additionally, it could lessen the extent of myocardial infarctions, lower the production of proinfammatory markers, and enhance heart function in mice [37].Several studies postulated that quercetin might prevent and treat COVID-19 due to its potent scavenger and anti-infammatory properties.Fifty COVID-19-infected individuals received a daily dosage of 1,000 mg of quercetin as part of a randomized clinical study [38].Various fruits contain Fisetin, which may be efective in preventing traumatic brain damage, schizophrenia, depressive disorders, stroke, and neuropathy associated with diabetes [39].Withaferin A has demonstrated anticancer activity in several cancer cell types, including multiple myeloma, neurological tumors, leukemia, and an aggressive form of ovarian, breast, and neck cancer [40].One of the approaches for determining the quality of herbal medication is phytochemical examination [41].Te ethanol extract of A. digitata (ADEE) bark revealed the presence of favonoids, phenols, triterpenoids, saponins, and other compounds.Previously, ADEE was reported on several disorders, such as cancer, renal, and diabetes.Te defense system against oxidation can remove reactive oxygen species (ROS), which 16 Scientifca have a crucial role in starting lipid peroxidation [42].DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity measures antioxidants hydrogen-donating ability, and the relative decrease in DPPH absorbance when it reacts with the antioxidant measures its activity [43].Te ethanol extract demonstrated a concentration-dependent increase in free radical trapping in the current investigation.In vitro, the ethanol extract lowered NO production in a concentrationdependent way.A superoxide anion is a reduced form of molecular oxygen formed by receiving one electron.
According to the fndings of this investigation, the ethanol extract has high superoxide quenching action.Tis could be related to the phenol level of the ethanol extract.Free radicals can harm macromolecules in cells like DNA, protein, and membrane lipids.Lipid peroxidation refers to the reaction of oxidative deterioration of polyunsaturated lipids.Peroxidation involves the direct reaction of oxygen and lipids to form radical intermediates and produces semiconstant peroxidases, damaging the enzymes, nucleic acids, membranes, and proteins [44].Te ethanol extract of bark prevents DNA damage by quenching free radicals in the current study.Te ferric thiocyanate (FTC) method was used to calculate the total antioxidant activity of bark and was compared to the thiobarbituric acid (TBA) method.Te antioxidant activity of ADEE was relatively high in this study.Tese points to the existence of antioxidant-active phytochemical elements like ascorbic acid and carotenoids both detected in ADEE.
Although there are multiple antibacterial agents to choose from, they often sufer from several disadvantages, such as low solubility, limited stability, high expense, poor bioavailability, challenges in accessing the site of action, frequent administration, signifcant side efects, and potential toxicity [45].At diferent concentrations, the antimicrobial activity of ADEE against Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Serratia marcescens, and Pseudomonas aeruginosa was observed.ADEE inhibited the growth of the test organisms at a concentration of 75 µg/ mL and 100 µg/mL with a slight zone of inhibition at 25 µg/ mL and 50 µg/mL.Tis antibacterial activity shown by ADEE indicated the active compound(s) extracted by the cold maceration method.P. aeruginosa was used to test pellicle production in glass tubes containing LB broth at two diferent temperatures (25 °C and 35 °C).After 24 hours, a thin pellicle formed on the media surface, followed by an opaque, strong, thick pellicle covering the whole media.Aerobic bacteria require this contact because they acquire oxygen from the air and nutrients from the liquid media [46].
Investigators distinguish closely related fora and fauna by applying the powerful tool FT-IR spectrum [47].In this present study, functional groups and bioactive compounds in the plant extract were revealed using FT-IR analysis.Revealing phytocompounds from the resultant GC-MS and FT-IR studies paved the way for analyzing the potential therapeutic phytocompounds.Te presence of functional groups such as ketones, carboxylic acid, alkanes, and silicon plays a key role in various medicinal ailments of A. digitata, in which silicon contributes to the proper functioning and prevents the toughening of arteries and veins.On the other hand, the contribution of carboxylic acid and hydroxyl groups leads to the proper functioning and metabolism, and the presence of such functional groups is considered a remarkable therapeutic ailment.Furthermore, computational studies revealed the phytochemicals' potency against the virulence factors of the targeted isolates and their antioxidant enzyme activity in this study.Previous studies reported that the phytocompounds such as apigenin and quercetin isolated from the bark extract possess antiplasmodial activity [48], whereas the interaction of quercetin and apigenin inhibit the β-hematin formation, which was revealed through the highest binding afnity and interaction of hydrogen bond formation.
To ascertain the strength of the interaction and determine the optimal orientation of the interaction between molecules present in Adansonia digitata and vascular endothelial growth factor receptor 2, which would result in a complex with protein with the least energy, molecular docking experiments have been carried out.All the compounds have demonstrated the suppression of hepatobiliary cancer-causing vascular endothelial growth factor 2 (VEGF2) [49].In this study, the lead phytocompounds showed better binding energy and interaction with the key amino acid residues easing the inhibitory efcacy against the target protein modeled pqsA and 2OXX.Residues such as Gly_302, Tyr_163, Gly_210, and Tr_87 from the modeled structure interacted with the hydroxyl and amine groups, facilitating the stronger interaction and structural stability of the docked complex.Results obtained from 2OXX interact with the phytocompounds explicitly showing its potent efcacy by forming hydrogen bonds and Pi-Pi interaction with the crucial residues with better binding afnity values.Te presence of ketone, carboxylic acid, alkanes, and polyhydroxy functional groups present in A. digitata illustrates the biological and pharmacological efcacy, while those functional groups play a crucial role in interacting with the key residues of the targets.
Research on the possibility of using extracts of plants to treat degenerative disorders that persist over time is rapidly expanding.Given the increasing frequency of these illnesses, the encouraging potential provided by plant extracts provides a glimpse of hope for people aficted with these devastating conditions.Te future perspective of this study deals with the phytocompounds from Adansonia digitata that could be examined against chronic diseases including cancer.Te overall study concluded that the ADEE showed stronger inhibition efcacy against the selected isolates through MIC and bioflm inhibition, while the studies were further evaluated by in silico approaches, which paves the way to conclude the potent drug-likening property of the phytocompounds present in Adansonia digitata.

Strengths and Limitations of the Study.
Tis investigation employs a multidisciplinary methodology and acknowledges the importance of conventional medicine as the origin of novel medicinal compounds.It pays attention to the commercial benefts that baobab trees ofer to rural areas.Despite their value, the computational investigations rely on simulations and predictions.Terefore, to confrm their accuracy and applicability, in vitro and in vivo research types are required.Furthermore, more investigation needs to be done, including clinical studies and quality control procedures, to verify its safety and efcacy for certain medicinal applications.A more comprehensive perspective can be provided by taking into consideration the cultural and economic facets of traditional medicine.According to the study's fndings, Adansonia digitata bark extract and its phytocompounds may be useful as medicines, especially against Pseudomonas aeruginosa and C2 kinase.

Conclusion
Adansonia digitata has been considered a versatile tree species used as a remedy against various illnesses, fodder, valued nutritional food, and regarded as raw material for making valuable items.Te outcome of the phytochemical screening analysis conducted during this investigation irrefutably confrms the presence of numerous phytoconstituents, such as alkaloid, saponin, favonoid, steroid, tannin, and terpenoids, in the stem bark extract.GC-MS and FT-IR spectroscopic analysis results show that the extract has numerous functional groups that contribute to its potential as a therapeutic.ADEE's ability to suppress pathogenic bacteria and its therapeutic efciency, especially in preventing the formation of pellicles and inhibiting bioflms, are probably due to a variety of secondary metabolites found in the bark.At lower concentrations, ADEE revealed more than 80% inhibition of P. aeruginosa bioflm formation while the observed MIC value of about 0.625 µg/mL.Utilizing the application of computational techniques to analyze the phytocompounds found in the bark of A. digitata, we have shown that this natural component possesses remarkable binding ability and constant stability against selected receptors.As a result, we can assert with certainty that the bark of A. digitata is a powerful source of therapeutic targets and therapeutic advantages, efciently alleviating a range of diseases.Additional investigation of these phytocompounds and their specifc measures may provide insightful information for conventional and modern applications.
Values are expressed as mean ± SD for triplicates.

Table 2 :
Antibacterial activity of Adansonia digitata ethanol extract (ADEE) through the agar well difusion method.
bcValues represented in the table are means of triplicate readings and standard error of the mean of zone of inhibition of isolates.a, b, c, and d likely represent statistically signifcant diferences between the zones of inhibition, which indicate the efectiveness of the extract against diferent bacteria at varying concentrations.

Table 3 :
Diferent concentrations of ADEE used for the MIC determination.Te value 0.625 represents the minimum inhibitory concentration (MIC) of the antibiotic tetracycline, indicating the lowest concentration (10 μg/mL) of the aqueous extract of ADEE at which tetracycline successfully inhibited bacterial growth.Tis MIC value highlights tetracycline's potency against the tested bacteria, even at a relatively low concentration when combined with ADEE.

Table 7 :
Binding free energy of the docked complexes computed by MM/GBSA.

Table 8 :
Toxicity prediction of the phytocompounds using ProTox-II server.