Molecular docking analysis and evaluation of the antibacterial and antioxidant activities of the constituents of Ocimum cufodontii

Ocimum cufodontii ((Lanza) A.J.Paton) has been traditionally used in Ethiopia against bacteria. The extracts of the leaves and roots of O. cufodontii after silica gel column chromatography furnished compounds 1–5, compounds 3 and 4 are new natural products. The oil from the hydro-distillation of the leaves, after analyzed with GC–MS, has led to the identification of β-caryophyllene as a principal component, suggesting the essential oil as medicine and spices to enhance the taste of food. The constituents of O. cufodontii were assessed for their antibacterial activity against E. coli, K. pneumonia, S. typhymurium and S. aureus. The best activity was displayed against S. aureus by the hexane extract of the roots, compound 4, and the essential oil with an inhibition zone of 17, 15, and 19 mm, respectively. Molecular docking analysis revealed that compound 1 has better docking efficiency and forms hydrophobic interactions with five amino acids (ARG192, PHE196, GLU185, GLU193, and LYS189). This suggests that the compounds may act as potential inhibitors of DNA gyrase. The constituents were also assessed for their antioxidant activities using DPPH, ferric thicyanate and ferric reducing power assay. The hexane extracts of the roots inhibited the DPPH radical and peroxide formation by 90.5 and 83%, respectively, suggesting the potential of the extract as an antioxidant. Furthermore, the hexane extract of the roots of O. cufodontii exhibited the maximum reducing power compared with the EtOAc and methanol extracts. Hence, the activity displayed herein indicated as the plant has great potential as a remedy for diseases caused by bacteria and radicals.

Extraction. Ground roots of O. cufodontii (300 g) were successively extracted on maceration with each 1.5 L of n-hexane, EtOAc and MeOH for 72 h following the standard procedure in the literature with slight modifications 16 . Each extracts was filtered and concentrated under vacuum using rotary evaporator at 40 °C to furnish 1.5, 1.2 and 3%, respectively. Likewise, the leaves (300 g) were also extracted following same procedure as above to afford 2, 1.5 and 4% of the n-hexane, EtOAc, and MeOH extract respectively.
Isolation of compounds. The EtOAc extract (3 g) of the leaves of O. cufodontii was adsorbed and fractionated over silica gel (150 g) column chromatography with n-hexane:EtOAc of increasing polarities as eluent to furnish 58 fractions, each 50 mL. Fraction eluted with n-hexane:EtOAc (7:3) gave compound 1 (40 mg).
Extraction of essential oil from the leaves. The ground leaves of O. cufodontii (60 g) were hydrodistilled in a Clevenger's apparatus for 4 h. The less dense yellowish oil separated using separatory funnel was dried over anhydrous Na 2 SO 4 , and kept in refrigerator until analysis. Experiments were conducted in triplicate. GC-MS analysis of the essential oil was carried out by following the method described in Hema 17 . A GC-MS instrument from Agilent Technologies (Santa Clara, CA, USA) equipped with a 6890 N network GC system, 5975 inert mass selective detector, 7683B series auto sampler injector (10 μL in size), G1701DA GC/MSD Chem Station and HP5MS column (30 m length × 0.25 mm internal diameter × 0.25 μm film thickness) coated with 5% phenyl 95% methyl poly siloxane was used for analyzing the samples. 2 μL essential oil solutions in chloroform was injected through auto sampler and analyzed with HP5MS column. Column temperature was programed as follows: 55-120 °C at 20 °C/min, 120-150 °C at 1.5 °C/min, 150-250 °C at 20 °C/min, 250 °C (10 min) and 3 min solvent delay. The mass spectra transfer line temperature was 280 °C. The carrier gas was helium (1 mL/min) with a split ratio equal to 100:1. The mass spectra were recorded in electron ionization mode at 70 eV with scanning from 50 to 500 amu (atomic mass unit) at 0.5 s with the mass source being set at 230 °C. The relative % amount of each component was calculated by comparing its average peak area to the total area Identification of the constituents was done with the aid of NIST 2005 library of mass spectra.

GC-MS analyses of the essential oil of the leaves of O. cufodontii.
Antibacterial activity. The antibacterial activity of the extracts and isolated compounds were tested using Mueller Hinton agar medium following previously reported protocol with slight modification 15  www.nature.com/scientificreports/ with a sterile swab moistened with the bacterial suspension and each 50 µg/mL of the extracts and isolated compounds were filled in the wells with the help of micropipette. Plates have been left for some time till the samples diffuse in the medium with the lid closed and incubated at 37 °C for 24 h. After overnight incubation, the plates were observed for the zone of inhibition (ZI) and the diameter of the inhibition zone was measured using scale. Then the samples were analyzed in triplicates and expressed as mean ± SD. Ciprofloxacin and DMSO were used as positive and negative controls, respectively.
Molecular docking studies of the isolated compounds. AutoDock Vina with previously reported protocol 17 was used to dock the proteins (PDB ID: 6F86) and isolated compounds (1)(2)(3)(4)(5) into the active site of proteins 18,19 . The chemical structures of compounds 1-5 were drawn using ChemOffice tool (Chem Draw 16.0) assigned with proper 2D orientation, and energy of each molecule was minimized using ChemBio3D. The energy minimized ligand molecules were then used as input for AutoDock Vina, in order to carry out the docking simulation. The crystal structure of receptor molecule E. coli gyraseB (PDB ID: 6F86) were downloaded from protein data bank. The protein preparation was done using the reported standard protocol 20 by removing the co-crystallized ligand, selected water molecules and cofactors. Next, the target protein file was prepared by leaving the associated residue with protein by using Auto Preparation of target protein file Auto Dock 4.2 (MGL tools1.5.6). The graphical user interface program 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 best docked 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 Discovery studio visualizer and PyMOL. The ligands are represented in different color, H-bonds and the interacting residues are represented in ball and stick model representation.
Antioxidant activity. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The radical scavenging activity of the samples were estimated using 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay 21 . Briefly, the methanol solution of the hexane extract was serially diluted with 0.004% DPPH in methanol to furnish 200, 100, 50, and 25 µg/mL of the extract. After 30 min incubation in an oven at 37 °C, absorbance at 517 nm was measured using UV-Vis spectrophotometer. Ascorbic acid and 0.004% DPPH in methanol were used as positive control and blank, respectively. The percentage inhibition of the extracts was calculated using the following formula. % Inhibition = (A control − A extract )/A control × 100 where Acontrol is the absorbance of 0.004% DPPH in methanol and Aextract is the absorbance of DPPH solution plus sample. The DPPH radical scavenging activity of the samples was also expressed as IC 50 , the concentration of the test compound to give a 50% decrease of the absorbance from that of the control solution. The radical scavenging activity of the other extracts and the isolated compounds of the leaves and the roots of O. cufodontii were also evaluated following the same. Ascorbic acid and quercetin were used as positive controls.
Ferricthiocyanate method. The lipid peroxidation inhibitory potential of the samples was evaluated using ferricthiocyanate method 22 . In brief, the hexane extract (0.1 mg) of the leaves of O. cufodontii, linoleic acid (100 µL), EtOH (5 mL) and phosphate buffer (5 mL, 0.05 M, pH = 7) in water were mixed and incubated at 40 °C in an oven. After 24 h, 0.1 mL was taken and added in to a vial containing 75% aqueous EtOH (7 mL), 30% of NH 4 SCN (0.15 mL) and 0.15 mL of 0.02 M FeCl 2 in 3.5% HCl. The absorbance of the red coloured solution was measured at 500 nm using UV-Vis spectrophotometer. This was repeated every 24 h until the control gave its maximal absorbance value. Likewise, the lipid peroxidation inhibitory activity of the EtOAc extract of the leaves, the hexane and EtOAc extracts of the roots, and isolated compounds were assessed following the same procedure. Absorbance of the blank and ascorbic acid was measured in the same fashion. The percentage inhibition using ferricthiocyante method has been calculated according to the following formula.
where As is absorbance of the sample and Ab is absorbance of the blank 23 .
Ferric-reducing antioxidant power (FRAP) assay. The ferric reducing antioxidant power assay of the extracts and isolated compounds were measured according to Birasuren et al. 24 . The FRAP reagent was prepared by mixing acetate buffer (25 mL, 0.3 M), TPTZ (2,4,6-tripyridyl-s-triazine) solution (2.5 mL), and FeCl 3 •6H 2 O solution (2.5 mL), and then heated to 37 °C before use. A properly diluted sample (0.1 mL, 100 µg/mL) was mixed with FRAP reagent (4.0 mL) to form a mixture. This was incubated at 37 °C for 10 min in the dark, and the absorbance was measured at 593 nm against a blank (distilled water). The results were expressed as aqueous solution of ferrous sulfate (FeSO 4 •7H 2 O), and derived from a calibration curve of the standards. Likewise, the FRAP value of ascorbic acid was obtained by the same procedure.
Statistics data analysis. The antibacterial and antioxidant assay data generated by triplicate measurements were reported using mean ± standard deviation. Analyses were performed using GraphPad Prism version 5.00 for Windows, (GraphPad Software, San Diego California USA, www. graph pad. com"). Groups were analyzed for significant differences using a linear model of variance analysis (ANOVA) test for comparisons, with level significance at p < 0.05 (Supplementary Information).

Results and discussion
Characterization of isolated compounds. In the present work, five compounds from the roots and leaves of O. cufodontii were isolated and characterized (Fig. 1). The structure elucidations of these compounds are described herein. Compound 1 (40 mg) was isolated as a white solid melting at 295-296 °C. TLC showed a spot at Rf 0.47 with n-hexane:EtOAc (7:3) as eluent which was similar to the one reported for betulinic acid 25  were due to methyl protons at C-23 and C-24, respectively. In addition, the spectrum also showed a multiplet at δ 2.93 (1H, m, H-19) and 1.50 (1H, m, H-18). These spectral features are in close agreement to those reported for betulinic acid 26 . The 13 C-NMR spectrum of compound 1 displayed the presence of 30 carbon signals of which six methyl, six quaternary, one carboxylic acid, eleven methylene, and six methine, confirming that compound 1 is betulinic acid 27 .
Compound 2 (32 mg) was isolated as a yellow solid from n-hexane extract of the root of O. cufodontii. The MS spectrum revealed the molecular ion peak m/z 345 [M + H] + attributed to the molecular formula C 21 H 29 O 4 . The UV-Vis spectrum (MeOH) showed absorption maxima at 140, 240 and 290 nm suggesting a quinone skeleton 28 . The 1 H-NMR spectral data revealed the presence of two methoxy signals δ 3.90 and 3.91. The absence of methine proton signals in the olefinic region justify that the carbons in this regions are all quaternary. The spectrum also displayed signals due to methylene protons at δ 1.  (Table 1) together with DEPT-135 NMR spectrum revealed the presence of seven quaternary, five methine, two methylene, and five methyl groups. The presence of three carbonyls are also evident at δ 213.6 (C-3″), 181.2 (C-1′) and 182.3 (C-4′). The two methylenes were observed at δ 24.9 (C-1″) and δ 38.5 (C-2″). The spectrum also displayed signals due to quaternary carbons at δ 147.0 (C-2), 144.7 (C-6), 134.9 (C-2′), and δ 128.4 (C-5). The signal due to two methine group on aromatic ring were evident at δ 128.5 (C-3) and δ 137.0 (C-4). The data generated enable us to propose the structure of compound 3 as shown in Fig. 1. Correlations are also observed in the HMBC spectrum due to the proton at δ 7.04 with the carbon at δ 140.2 and 128. The proton at δ 7.34 correlates with the carbon at δ 147, 134, and 19. Therefore, compound 3 is a novel compound isolated for the first time from the roots of O. cufodontii with its structure depicted in Fig. 1.   Antibacterial activity. The findings of the in vitro antibacterial activity of the extracts and isolated compounds from the leaves and roots of O. cufodontii were presented in Table 3.
As clearly depicted in Table 3   Antioxidant activity. DPPH assay is a simple method to assess antioxidants activities by measuring absorbance at 517 nm due to the formation of DPPH radical 29,31 . The extracts and isolated compounds from the leaves and roots of O. cufodontii change the purple colored DPPH radical to the yellow-colored diphenylpicrylhydrazicine. The extracts also reduce the absorbance of the DPPH radical at 517 nm indicating their potential as radical scavengers. The activity increases in a dose dependent manner. The hexane extract of the roots of O. cufodontii reduced the DPPH radical by 90.50% at 200 µg/mL (Table 4). This is superior to the activity displayed by the other extracts. This is evident from the low IC 50 (4.4 µg/mL) value observed for the hexane extract while the IC 50 value of the hexane extract of the leaves, the EtOAc extract of the leaves and the roots were 6.0, 48 and Table 3. Inhibition zone (in mm) displayed by constituents of the leaves and roots of O. cufodontii at dose of 50 µg/mL. Samples were analyzed in triplicates and expressed as m ± SD. HLE hexane leaves extract, EALE EtOAc leaves extract, MLE MeOH leaves extract, HRE hexane root extract, ERE EtOAc root extract, MRE MeOH root extract, EO Essential oil.    Table 4, the DPPH radical scavenging activities of the isolated compounds are inferior compared with the activity shown by extracts. This is likely ascribed to the presence of phenols and flavonoids in the extracts of the roots and leaves of O. cufodontii. The presence of flavonoids was further confirmed by the formation of yellow orange color on treating the extracts of the roots and leaves of O. cufodontii with NaOH followed by HCl 34 . Moreover, the extracts furnished a bluish green color on treatment with FeCl 3 supporting the presence of phenols in the extracts 35 .
The lipid peroxidation inhibitory potential of the extracts and isolated compounds were assessed using ferricthiocyante methods with the results presented in Table 4. As observed from Table 4, the hexane extracts of the leaves and the roots inhibited peroxide formation by 70.01 and 83.02%, respectively. The activity displayed by the latter extract is better than the activity displayed by the isolated compounds. The activity displayed by the hexane extract of the roots of O. cufodontii is comparable with the standard drug demonstrating the lipid peroxidation inhibitory potential of the hexane extract. This indicates the potential use of the extracts of the leaves and roots of O. cufodontii as remedy against diseases caused by radicals.
Ferric-reducing antioxidant power (FRAP) assay. FRAP assay is used to evaluate the antioxidant potential of extracts based on the reduction of the Fe 3+ -TPTZ (2,4,6-tripyridyl-s-triazine) complex to the ferrous form at a pH around 3.6 24 . The ferric-TPTZ complex can be monitored at 593 nm 36 . In view of this, the ferricreducing antioxidant power of the extracts and isolated compounds was assessed and the results are depicted in Table 5.
The higher absorbance of the extract indicates a higher ferric reducing power. As clearly seen from

Molecular docking studies.
Compounds with antimicrobial activities disable bacteria by targeting key components of bacterial metabolism including cell-walls, DNA-directed RNApolymerase, protein synthesis, enzymes and DNAgyrase. The latter controls the topology of DNAduring transcription and replication by introducing transient breaks to both DNA strands [37][38][39] . Since DNAgyrase is pivotal for bacterial survival, it is essential to exploits bacterial DNAgyrase as a key target of antibacterial agent 39 . In view of this, the molecular docking study was carried out to examine the binding interactions of isolated compounds with the pocket of DNAgyrase and compared with ciprofloxacin. All the synthesized compounds exhibited well established bonds with one or more amino acids in the active pocket of the enzyme. The compounds also displayed minimum binding energy ranging from − 6.1 to − 6.9 kcal/mol ( Table 6) with compound 3 shown to have comparable binding score and amino acid interactions compared to ciprofloxacin. The binding affinity of the synthetic compounds along with their bonding interactions of ligands (1)(2)(3)(4)(5) were summarized in Table 6.
The extracted natural product molecules 1-5 showed better docking efficiency with DNA gyrase B within the binding pocket. In comparison to ciprofloxacin, all the isolated compounds have shown similar residual amino acids binding interactions with Ala-47, Glu-50, Gly-77, Ile-78, Pro-79, Ile-94, Thr-165 (Hydrophobic) and Asp-73, Arg-76, Asn-46 (Hydrogen bonds). The, isolated compound 1 forms additional residual interaction with Val-120 in the active site of the target protein with the docking score − 6.3 kcal/mol. Compound 2 has the docking score − 6.1 kcal/mol with no hydrogen bond or additional residual interactions within the binding pocket. The compounds 3 and 4 each formed additional residual Van dar Waals interaction with residual amino Table 5. Ferric-reducing potential of extracts and isolated compounds of O. cufodontii. Samples were measured in triplicates and expressed as M ± SD; Ascorbic acid was used as positive control.

Conclusion
In conclusion, two novel compounds 3 and 4 have been isolated and characterized from the roots extracts of O. cufodontii. The n-hexane extracts of the roots, the essential oil of the leaves of O. cufodontii, and compound 4 have shown comparable results to ciprofloxacin. The radical scavenging activity and anti-lipid peroxidation potential displayed by the n-hexane extracts of the roots of O. cufodontii also suggests the potential of the plant as an antioxidant. The in silico molecular docking studies revealed that all the isolated compounds 1-5 have close binding energy to the standard drug and may be considered as a good inhibitor of DNA gyrase. Hence, the activity displayed herein indicates that the plant has a great potential as a remedy for diseases caused by bacteria and radicals. Furthermore, the results obtained in the present study may help substantiate the traditional use of the roots of O. cufodontii with modern scientific based medical treatment.