Natural Products Chemistry & Research

Molecular Docking Analysis and Evaluation for Antibacterial Activity of Chemical Constituents From the Stem Barks of Podocarpus falcatus

Desalegn Abebe Deresa^* and Negera Abdissa ^*Correspondence: Desalegn Abebe Deresa, Department of Chemistry, College of Natural and Computational Sciences, Wollega University, Nekemte, Ethiopia, Email:

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Introduction

Medicinal plants have been used in the treatment of various diseases sinThe Podocarpus (family, Podocarpaceae) is one of the largest genera of all conifers of the family containing about 94 species distributed from south temperate zones through the tropical highlands, West India, and Japan. Species from this genus have been reported to produce cytotoxic nor- and bisnor-diterpenoid dilactones generally known as nagilactones or podolactones [1]. In addition, totarane-type diterpenes such as totarol and their dimers (macrophyllic acid) have also been reported from many species of Podocarpus. These compounds are considered to be chemical markers of the genus [2]. Podocarpus falcatus (Thunb.) belongs to the vernacular name zigiba. It is an evergreen, dioecious, medium to largesized tree up to 60 m tall and widely distributed in Ethiopia, Kenya, Tanzania, Mozambique, South Africa, and Madagascar [1, 2]. Besides its commercial and ecological importance, it has also known for its ethnobotanical values in Ethiopia. The roots of this plant are used as anticancer remedies [3,4]. Crushed and juice of the leaves taken for vomiting, whereas the root dried powdered, mixed with water taken for febrile illness. Decoction of the fruit serves as a tonic for cleaning the kidneys, lungs, and stomach [5 ]. Despite the wider use of this plant by the communities for medicinal purposes, the phytochemical and bioactivity information about the stem bark of this plant is limited. Therefore, as part of the search for new bioactive molecules from Ethiopian medicinal plants, the isolation of five compounds and antibacterial activities of the extract and the compounds were reported here.

Materials and Methods

General method

Solvents and reagents used for extraction and purification of the compounds are of analytical and HPLC grade. Analytical TLC pre-coated sheets ALUGRAM®Xtra SIL G/UV₂₅₄ (layer: 0.20 mm silica gel 60 with fluorescent indicator UVF_254/365) were used for purity analysis. For column chromatography, silica gel 100 mesh-200 mesh was used. Chromatograms were visualized on TLC by spraying with 10% H₂ SO₄ and heating on a hot plate. NMR spectra data were recorded on an Avance 600 MHz spectrometer (Bruker, Billerica, MA, USA, at 600MHz (1 H) and 150MHz (13C). Chemical shifts are expressed in ppm, and J values are given in Hz and referenced to the central peak of the appropriate deuterated solvent's resonances (residual CDCl₃ , (CD₃ ) ₂ CO, MeOD, and (CD₃ ) ₂ SO at δH₇.26, 2.20, 3.35, 2.52 for protons and δC 79.16, 205.87, 49.77, 40.76 for carbons respectively). Whatman filter paper No.3, DMSO, Petri dishes, and gentamycin were used in the antibacterial analysis.

Plant materials

The stem bark of P. falcatus was collected from Horro Buluk, Horro Guduru Wollega zone, Oromia regional state, Ethiopia in September 2020. The plant material was identified by botanist (Dr. Fekadu Gurmessa) and the voucher specimen (DAD002Pf) has been deposited in Wollega University Herbarium. The collected plant part was washed thoroughly with tap water and cut into smaller pieces and dried under shade.

Extraction and isolation

The powdered stem bark of P. falcatus (850 gm) was extracted with an equal ratio of CH₂ Cl₂ -MeOH (3 Lx₃ L) at room temperature for 48 hours each with occasional shaking. The crude extract was filtered from marc using Whatman filter paper. The solvent was evaporated under reduced pressure using a rotary evaporator at 400 C to yield (20 gm, 2.7%) of dark brown crude extract. About 18 g of the extract was adsorbed on 25 gm of silica gel and subjected to column chromatography, packed with silica gel (320 g). The column was eluted with hexane with an increasing gradient of ethyl acetate to afford 30 major fractions ca. 100 mL each. Fractions with similar TLC profiles were combined for further purification. Fractions 6–10 (2% EtOAc in hexane) were combined 20 mg and refined by Sephadex LH₂0 (eluting with CH₂ Cl₂ /MeOH; 1:1) afforded (1 mg, 15 mg) while fractions 15-20 (3% EtOAc in hexane) showed similar TLC profiles were combined gave 18 mg and purified by washing with excess petroleum ether gave compound (2 mg, 12 mg). Fractions 26-30 (6% EtOAc in hexane) showed similar spots combined afforded 15 mg and purified further by Sephadex LH-20 (eluting with CH₂ Cl₂ /MeOH; 1:1) afforded compound (3 mg, 10 mg) and compound (4 mg, 8 mg).

The powdered stem bark of P. falcatus (850 gm) was extracted with an equal ratio of CH₂ Cl₂ -MeOH (3 Lx3 L) at room temperature for 48 hours each with occasional shaking. The crude extract was filtered from marc using Whatman filter paper. The solvent was evaporated under reduced pressure using a rotary evaporator at 400 C to yield (20 gm, 2.7%) of dark brown crude extract. About 18 g of the extract was adsorbed on 25 gm of silica gel and subjected to column chromatography, packed with silica gel (320 g). The column was eluted with hexane with an increasing gradient of ethyl acetate to afford 30 major fractions ca. 100 mL each. Fractions with similar TLC profiles were combined for further purification. Fractions 6–10 (2% EtOAc in hexane) were combined 20 mg and refined by Sephadex LH₂0 (eluting with CH2 Cl2 /MeOH; 1:1) afforded (1 mg, 15 mg) while fractions 15-20 (3% EtOAc in hexane) showed similar TLC profiles were combined gave 18 mg and purified by washing with excess petroleum ether gave compound (2 mg, 12 mg). Fractions 26-30 (6% EtOAc in hexane) showed similar spots combined afforded 15 mg and purified further by Sephadex LH-20 (eluting with CH₂ Cl₂ /MeOH; 1:1) afforded compound (3 mg, 10 mg) and compound (4 mg, 8 mg).

Antibacterial activity assay

Five pathogenic bacterial strains, one gram-positive (Staphylococcus aureus (ATCC25923)) and four gram-negative (Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella typhimurium (ATCC13311), and Shigella flexneri (ATCC29903) were obtained from the Department of Biology, Wollega University and used for evaluation of antibacterial activities. Antibacterial activities of the methanol extract and isolated compounds were tested against five bacterial strains using the disc diffusion method [6] with slight modification. The test solutions were prepared with a known weight of crude extract (0.01 mg) and isolated compounds were dissolved in 1 mL of DMSO. A 0.6 mm diameter sterile Whatman test disk was placed on the surface of the inoculated Mueller Hinton Agar in a 90 mm Petri dish and then 0.01 mg/ mL of the crude extract and the isolated compound 1-3 were applied onto the disks. Gentamicin (10 µg) and DMSO were used as the positive and negative controls respectively. The test samples were allowed to diffuse for 30 min and the plates were then kept in an incubator at 37°C for 24 hours [7]. The experiments were carried out in triplicate and the mean diameter of the inhibition zones was calculated. The antibacterial activity was determined by measuring the zone of growth inhibition surrounding the disks Antibacterial inhibition activities were measured against the standard.

In silico molecular docking methodology

Preparation of ligands

The 2D structures (.mol) of all isolated compounds 1-3 were drawn and each structure was analyzed by using ChemDraw 16.0. The selected molecules were treated quantum mechanically by applying the DFT method using the Gaussian 09 program suite at the Becke-3-Lee-YangPar (B3LYP) level combined with the standard 6-31G (d,p) basis set. During the optimization procedure, all the parameters were set to obtain a stable structure with minimum energy. The global minimum energy of the title compound was determined from the structure optimization procedure. The 3D coordinates (PDB) of each molecule were obtained through the optimized structure.

Autodock Vina analysis

The graphical user interface program Auto Dock 4.2.6 was used to set the grid box for docking simulations. The grid was set so that it surrounds the region of interest in the macromolecule. The docking algorithm provided with Auto Dock Vina was used to search for the bestdocked conformation between ligand and protein. During the docking process, a maximum of nine conformers were considered for each ligand. The conformations with the most favorable (least) free binding energy were selected for analyzing the interactions between the target receptor and ligands by the Discovery studio visualizer. AutoDock Vina with the standard protocol was used to dock the protein S. aureus Gyrase (PDB ID 2XCT) and human topoisomerase IIβ with DNA (PDB ID 3QX3) and isolated ligands 1-3 into the active site of proteins. The molecular docking studies were carried out using Auto Dock Tools (ADT) [8], which is a free Graphical User Interface (GUI) for the AutoDock Vina program. The grid box was constructed using 20x20x20, pointing in x, y, and z directions, respectively, with a grid point spacing of 0.375 Å. The center grid box is 62 Åx30 Åx62 Å for 2XCT and of 65 Åx40 Åx65Å for 3QX3. Nine different conformations were generated for each ligand scored using AutoDock Vina functions and were ranked according to their binding energies. The ligands are represented in a different color, H-bonds, and the interacting residues are represented in stick model representation.

Result and Discussion

The stem bark of P. falcatus was exhaustively extracted with an equal ratio of CH₂ Cl₂ -MeOH solvent combination. The extract was subjected to column chromatography on silica gel followed by purification on Sephadex LH-20 and afforded three compounds 1-3 (Figure 1).

Figure 1: Structure of isolated compounds from stem bark of P. falcatus

Characterization

Compound 1 was isolated as a white powder with melting points of 1750 C-1770 C. The¹ HNMR spectrum showed 12 signals, with a highly downfield shifted proton signal at 12.07 assigned to carboxylic acidic proton whereas, proton signals at 6.96 (1H, d, 8.6 Hz) and 6.41 (1H,d, 8.6 Hz) assigned to ortho-coupled aromatic protons (H-11, H-12), two overlapped doublets at 1.33 (6H, d, 7.1 Hz) were an isopropyl moiety and the rest proton signals correspond to non-aromatic protons. 13C NMR spectrum showed six aromatic carbon signals resonating at δC 149.5, 142.1, 134.6, 133.4, 126.6 and 113.4 assigned to C-13, C-9, C-14, C-8, C-11, and C-12 carbons, respectively. The remaining protons and carbons were assigned based on 2D-NMR data, notably, HSQC and HMBC. The COSY spectrum showed coupling between H-2/H-3 and H-5/H-6. The HSQC spectrum showed the presence of twelve signals suggesting that compound 1 possesses four saturated methylene groups, although their respective proton signals could not be fully determined due to their significant overlapping. The HMBC correlations between methyl protons H-16 and H-17 with C-14 indicated that the isopropyl group is attached to the aromatic ring at C-14. Moreover, a cross peak in the HMBC spectrum between H-18 and δC at 178.9 ppm, confirmed the presence of a carboxylic acid group attached at C-4. Similarly, C-4 at δC 43.4 was assigned based on an HMBC cross peak to H-18 (Figure 2). The above evidence was in agreement with a totarane-type diterpenes skeleton and corresponded to the known compound 4β-carboxy-19-nortotarol, which matched with the reported data for this compound [9].

Figure 2: The 2D and 3D binding interactions of compounds (1-3) and Reference drugs Doxycycline and Ciprofloxacin against S. aureus Gyrase (PDB ID: 2XCT).

Compound 2 was isolated as a white powder with melting points of 1340 C–1360 C. The structure of this compound was identified to be β-sitosterol using 1 H and ¹³C spectra data. The¹ H NMR spectrum showed an olefinic proton at δ_H 3.54 (1H, tdd, 11.2, 6.5, 4.6 Hz) corresponding to H-6 and oxymethine proton at δ_H 3.54 (1H, tdd, 11.2, 6.5, 4.6 Hz) for H-3. It also showed proton signals at δH 0.69 (3H, s), 1.02 (3H, s), 0.94 (3H,d, 6.5 Hz), 0.84 (3H, d, 6.8 Hz), 0.81(3H, d, 6.8 Hz), 0.85 (3H, t, 7.2 Hz) for six methyl groups and were assigned to H-18, H-19, H-21, H-26, H-27 and H-29, respectively. The ¹³C NMR spectrum showed signals for 29 carbon atoms including signals for six methyl (19.8, 19.4, 19.1, 18.8, 11.9 and 11.8), eleven methylene (δ_C 42.2, 39.8, 37.3, 33.9, 31.9, 31.6, 28.3, 26.1, 24.3, 23.1 and 21.1), nine methine (^δC 121.7, 71.8, 56.8, 56.1, 50.1, 45.8, 36.2, 31.9 and 29.2) and three quaternary (_δC 140.7, 42.3 and 36.5) carbon atoms. The recognizable signals at 140.9 (C-5) and 121.9 (C-6) are typical alkenes double bonds. The signals at δ 19.2 and 12.1 correspond to angular methyl carbon atoms (C-19) and (C-18) respectively . Signal at 71.9 is assignable to the β-hydroxyl group attached to the carbon at (C-3). Therefore, based on these spectral data agrees with existing literature reported on β-sitosterol [10].

Compound 3 was isolated as white amorphous with melting points of 2140 C-2150 C. The 1 H-NMR spectrum showed four proton signals. Signals atδ_H 7.78 (2H, d, 8.5Hz) were assigned to two overlapping aromatic protons (H-2, H-6), and signals at 6.82 (2H, d, 8.6Hz) were allocated to two overlapping protons (H-3, H-5). Whereas proton signals at 12.41(1H; s) correspond to a carboxylic acid proton, and signal at 10.22 (1H, s) is assigned to an OH proton. The ¹³C NMR spectrum showed seven carbons signals corresponding to four aromatic methine at ^δC 131.9 assigned to two overlapping carbons (C-2, C-6) and at 115.6 assigned to two overlapping carbons (C-3, C-5), three quaternary, one for carboxylic acid at 167.6 (C7),121.8 (C-1) and 162.1(C-4) . The 2D experiment COSY and HSQC spectra of compound 3 allowed, respectively, the detection of the scalar couplings of the protons and connectivity of each proton to a directly linked carbon atom. The COSY spectrum shows the coupling between H-2/ H-3 and H-5/H-6.The HSQC shows two protonated carbons at 7.79 (H-2 and H-6) linked with 131.9 (C-2 and C-6) and 6.82 (H-3 and H-5) linked with 115.6 (C-3 and C-5). The HMBC spectrum reveals the correlation between the cross peak ^δH 7.79 (H-2and H-6) correlated with 115.6 (C-3and C-5), 131.9 (C-4) and 167.6 (C-7), the proton at 6.82 (H-3 and H-5) correlated with 121.8 (C-1), 115.6 (C-3), 162.1 (C-4) and proton at 10.22 (OH proton) correlated with 115.6 (C-3 and C-5) and 162.1(C-4) that reveals the position of OH at C-4. Based on spectral analysis of 1D and 2D NMR compound 3 was identified as 4-hydroxybenzoic acid which is in agreement with reported data for 4-hydroxybenzoic acid [11].

Antibacterial activity result

The antibacterial activity of the crude extract and the isolated compounds result (Table 1) showed varying degree of inhibition of the growth of bacterial strains. The crude extract showed considerable activity on both Gram-positive and Gram-negative bacterial strains with a zone of inhibition ranging from 18.23 mm ± 0.21 mm-23.03 mm ± 0.05 mm with the highest activity (23.03 mm ± 0.05 mm) was observed against S. aureus, which is even greater than that of the reference drug (gentamycin, 19.5 mm ± 0.04 mm) against the same strain Whereas, the isolated compounds showed moderate activities against all the test strains. Compound 3 displayed strong activity against S. aureus (18.13 ± 0.12 mm) and compound 2 showed better inhibition zone against S. typhimurium (18.01 mm ± 0.47 mm). This variation in inhibition of the bacterial growth by the crude extract and isolated compounds could be related to the synergetic effects of the various kinds of compounds present in the crude extracts or the minor compounds in the extract that could showed this activity have not been isolated. Therefore, the antibacterial activity displayed by the stem extracts of P. falcatus reveals the presence of compound 1-3. In general, the remarkable activities of the crude extract from this medicinal plant (P. falcatus) support the traditional use of the plant and could be used as a potential candidate in the development of novel antibacterial agents.

Table 1. Antibacterial activity test for crude and isolated compounds from P. falcatus.

*PfME- Podocarpus falcatus Methanol Extract

Molecular docking analysis

The results obtained from the molecular docking study revealed that the isolated compounds 1-3 showed a strong binding affinity towards S. aureus Gyrase (PDB ID 2XCT) with binding energy values ranging from −6.1 kcal/mol to −8.6 kcal/mol, concerning ciprofloxacin and Doxycycline −8.4 kcal/mol and 13.0 kcal/ mol respectively (Table 2 and Figure 2). Compound 1 showed strong binding energy (−8.6 kcal/mol) compared to ciprofloxacin −8.4 kcal/mol and compounds 2 and 3 had good binding energy (-7.5 kcal/ mol, -6.1 kcal/mol) respectively compared to ciprofloxacin (−8.4 kcal/ mol). Compounds 1 and 2 showed one hydrogen bond each, compound 1 via an acid functional group with Arg-458 and compound 2 through its hydroxyl functional group with Met-1121 protein residues. Compound 3 displayed 5- Hydrogen bonds including 3 via its acid functional group with (DA-13, DG-9and DT-8) and 2 through its hydroxyl functional group with Arg-1122 and Gly-1082 protein residues. Hydrophobic interactions were observed for all isolated compounds 1-3 suggesting the compounds act as inhibitors of S. aureus Gyrase. On the other hand, the isolated compounds 1 and 2 showed a strong binding affinity towards Human topoisomerase IIβ with binding energy values ranging from −9.2 kcal/mol to −10.1 kcal/ mol, for Vosaroxin −10.2 kcal/ mol (Table 3, Figure 3). Compound 3 showed weak binding energy ranging from −6.4 kcal/mol compared to Vosaroxin (−10.2 kcal/mol). Compound 1 displayed 3-Hydrogen bonds 2 via its acid group with DC-11 and DG-10 and one trough hydroxyl group with Arg503 protein residues. Compound 2 showed 2- Hydrogen bonds through its hydroxyl group with Glu-477 and Asp-557 protein residues and compound 3 displayed 5-Hydrogen bonds including 3 via its acid functional group with Arg-503, DG-13, DC-8, and 2 through its hydroxyl group with Asp479 and Gly-478 protein residues. The result obtained suggests that compounds 1-3 are potential topoisomerase IIβ inhibitors and might be used as anticancer agents.

Table 2. Molecular docking results of isolated compounds against S. aureus Gyrase (PDB ID 2XCT).

DA= Deoxyadenosine; DG=Deoxyguanosine; DT=Deoxythymidine; DC=Deoxycytidine

Table 3. Molecular docking results of isolated compounds against Human topoisomerase II β (PDB ID 3QX3).

DA=Deoxyadenosine; DG = Deoxyguanosine; DT=Deoxythymidine; DC=Deoxycytidine

Figure 3: The 2D and 3D binding interactions of compounds (1-3) and reference drugs Abiraterone and Vosaroxin against human topoisomerase IIβ (PDB ID: 3QX3).

In silico pharmacokinetics (drug likeness) and toxicity analysis

The structures of isolated compounds 1-3 were converted to their canonical Simplified Molecular-Input Lineentry System (SMILE) and submitted to the Swiss ADME tool to estimate in silico pharmacokinetic parameters (drug-likeness properties) according to ‘Lipinski’s rule of five [12]. Lipinski’s rule of five implies that the drugs and/or candidates should obey the five-parameter rule, which states that Hydrogen-Bond Donors (HBDs) should be less than 5, Hydrogen-Bond Acceptors (HBAs) should be less than 10, molecular mass should be less than 500 Da, log P should not be less than 5, and Total Polar Surface Area (TPSA) should not be greater than 140Å. Drug-likeness is a prediction that screens whether a particular organic molecule has properties consistent with being an orally active drug [12]. In the present study, the Swiss ADME prediction revealed that compounds 1-3 obeyed Lipinski’s rule of five and they are likely to be orally active (Table 4). The TPSA value of the compounds 1-3 was noticed in the range from 20.23 Å to 57.53 Å and is well below the limit of 140 Å. The calculated numbers of rotatable bonds NRB values for the isolated compound 1-3 are less than 10 (Table 4), which indicated the compounds are conformationally stable.

Table 4. Drug-likeness predictions of compounds, computed by Swiss ADME.

NHD=Number of Hydrogen Donors, NHA=Number of Hydrogen Acceptors, NRB=Number of Rotatable Bonds, and TPSA=Total Polar Surface Area

ADMET properties

The Absorption, Distribution, Metabolism, Excretion, And Toxicity (ADMET) studies of isolated compounds 1-3 were predicted using Swiss ADMET. The skin permeability value (Kp) in cm/s indicates the skin absorption of molecules. In silico, the skin permeability, Kp, values of all compounds ranged from −2.20 cm/s to –6.02 cm/s suggesting low skin permeability and are within the range of broad-spectrum antibiotic Doxycycline (−9.03 cm/s) and under the clinical trial anticancer agent Vosaroxin (−8.98 cm/s). Additionally, Gastrointestinal (GI) and Blood– Brain Barrier (BBB) permeation indicate the absorption and distribution of drug molecules. The in-silico prediction results of Absorption, Distribution, Metabolism and Excretion (ADME) of the compounds 1-3 studied are presented in (Table 5). The Swiss ADME prediction parameters indicated that compound 1 and 3 showed high gastrointestinal (GI) absorption, whereas compound 2 displayed low absorption and do not show Blood– Brain Barrier (BBB) permeation. Moreover, a range of cytochromes (CYP’s) regulates drug metabolism, in which CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 are vital for the biotransformation of drug molecules [13]. Thus, in silico SwissADME prediction, only compound 1 inhibited cytochrome CYP2C9. However, the compounds 2 and 3 are neither cytochromes inhibitor nor a substrate of permeability glycoprotein (P-gp).

Table 5. ADME predictions of compounds, computed by SwissADME and PreADMET.

GI=Gastrointestinal, BBB=Blood Brain Barrier, P-gp=P-Glycoprotein, and CYP=Cytochrome-P

Acute toxicity prediction results, such as toxicity class classification and LD₅₀ values, predict that all of the isolated compounds 1-3 have no acute toxicity. The toxicological prediction gives results of endpoints such as hepatotoxicity, carcinogenicity, mutagenicity, and cytotoxicity. The studied compounds were predicted to be non- hepatotoxic, non- cytotoxic, non-mutagenic and non-irritant. However, compound 2 is immunotoxic as shown in (Table 6).

Table 6. Toxicity prediction of compounds, computed by ProTox-II and OSIRIS property explorer.

Conclusion

Phytochemical investigation of DCM-MeOH extract of stem bark. P. falcatus led to the isolation of four compounds 4β-carboxy-19-nor-totarol (1), β-sitosterol (2) and4-hydroxybenzoic acid (3), and their structures were established on the basis of their 1 H and 13C NMR spectral data and comparing with existing literature. Compound 3 is reported for the first time from the genus Podocarpus. The crude extract showed strong activity against S. aureus. Whereas, the isolated compounds showed moderate activity against all test strains. In silico molecular docking and ADMET analysis of isolated compounds 1-3 revealed that these compounds could be considered as antibacterial and anticancer agents. The results obtained from molecular docking, drug-likeness properties, ADMET analysis, in good agreement with those obtained from experimental studies suggesting the potential use of the isolated compounds as potential drug leads, which corroborate with the traditional uses of the roots of P. falcatus. Further comprehensive evaluations including in vivo activity tests could be done for conclusive decision on potential candidacy of the plant for formulation and medicinal uses.

Conflict of Interests

The authors declare that they have no conflict of interest.

Availability of Data and Materials

NMR data of compounds are available.

Acknowledgements

Mr. Desalegn A. is thankful to Wollega University, Ethiopia for material and financial support for his PhD study

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