Molecular docking of bioactive compounds extracted and purified from selected medicinal plant species against covid-19 proteins and in vitro evaluation

Bioactive compounds are secondary metabolites of plants. They offer diverse pharmacological properties. Peganum harmala is reported to have pharmaceutical effects like insecticidal, antitumor, curing malaria, anti-spasmodic, vasorelaxant, antihistaminic effect. Rosa brunonii has medicinal importance in its flower and fruits effective against different diseases and juice of leaf is reported to be applied externally to cure wounds and cuts. Dryopteris ramosa aqueous leaf extract is used to treat stomach ulcers and stomachaches. Each of these three medicinal plants have been indicated to have anticancer, antiviral, antioxidant, cytotoxic and antifungal effects but efficacy of their bioactive compounds remained unexplored. Study was aimed to explore In-vitro and In-silico anticancer, antiviral, antioxidant, cytotoxic and antifungal effects of bioactive compounds of above three medicinal plants. DPPH and ABTS assay were applied for assessment of antioxidant properties of compounds. Antibacterial properties of compounds were checked by agar well diffusion method. Brine shrimp lethality assay was performed to check cytotoxic effect of compounds. Molecular docking was conducted to investigate the binding efficacy between isolated compounds and targeted proteins. The compound isomangiferrin and tiliroside presented strong antioxidant potential 78.32% (± 0.213) and 77.77% (± 0.211) respectively in DPPH assay while harmaline showed 80.71% (± 0.072) at 200 µg/mL in ABTS assay. The compound harmine, harmaline and PH-HM 17 exhibited highest zone of inhibition 22 mm, 23 mm, 22 mm respectively against Xanthomonas while Irriflophenone-3-C-β- D-glucopyranoside showed maximum zone of inhibition 34 mm against E. coli. The compound isomangiferrin and vasicine contained strong antibacterial activity 32 mm and 22 mm respectively against S. aureus. The compound mangiferrin, astragalin, tiliroside, quercitin-3-O-rhamnoside showed maximum inhibitory zone 32 mm, 26 mm, 24 mm and 22 mm respectively against Klebsiella pneumoniae. Highest cytotoxic effect was observed by compound tiliroside i.e. 95% with LD50 value 73.59 µg/mL. The compound tiliroside showed the best binding mode of interaction to all targeted proteins presenting maximum hydrophobic interactions and hydrogen bonds. The binding affinity of tiliroside was − 17.9, − 14.9, − 14.6, − 13.8, − 12.8 against different proteins 6VAR, 5C5S, IEA3, 2XV7 and 6LUS respectively. Bioactive compounds are significant natural antioxidants, which could help to prevent the progression of various diseases caused by free radicals. Based on molecular docking we have concluded that phytochemicals can have better anticancer and antiviral potential.


Results and discussion
New therapeutic compounds can be synthesized using secondary metabolites found in medicinal plants.The types of secondary metabolites in solvent extract may vary depending on the solvent and extraction method.Different compounds like harmine, harmaline, vasicine, vasicinone (P.harmala) mangiferrin, isomangiferrin, irriflophenone -3-C-β-D D-glucopyranoside (D. ramosa) tiliroside, astragalin and quercitin-3-O rahmnoside (R. brunonii) were subjected for evaluation of their antioxidant, cytotoxic and antimicrobial potential.Molecular docking of these compounds was also done against different diseases (cancer, hepatitis B virus, influenza virus and SARS Covid 19).
DPPH and ABTS assays were used to check antioxidant potential.In DPPH assay all compounds showed significant free radical scavenging potential but the compound isomangiferrin and tiliroside exhibited highest antioxidant potential of 78.32% (± 0.213) and 77.77% (± 0.211) at 200 µg/mL (Fig. 1).In term of IC 50 values isomangiferrin showed IC 50 value of 93.85 µg/mL and tiliroside showed IC 50 value 78.77 µg/mL that were less than ascorbic acid showed IC 50 value 97.065 µg/mL.The low IC 50 means strongest scavenging potential.It means that scavenging capacity of these compounds were strongest than ascorbic acid.Quercitin-3-O rhamnoside exhibited lowest 50% free radical scavenging potential with IC 50 value 185.27 µg/mL concentration (Table 1).The IC 50 value of compounds was calculated by linear regression equation (Table 1).The dark violet color changed to light yellow also predicted antioxidant potential of compounds.Our study is in correspondence to a previously conducted study in which isomangiferrin and tiliroside showed strongest antioxidant potential in DPPH assay as compared to other compounds 15,21,24,25 .
Different compounds showed different inhibitory zones against different bacterial strains.Previously pharmacological actions of P. harmala including anti-oxidant, anti-tumor, hypoglycemic effect, leukemia healing, anti-inflammatory and analgesic properties, cytotoxic anti-tumor, and anti-microbial effects has been reported by Asgarpanah and Ramezanloo 29 .In present study the compound harmine and harmaline exhibited highest zone of inhibition 22 mm and 23 mm respectively against Xanthomonas while vasicine showed 22 mm against S. aureus.Irriflophenone-3-C-β-D-glucopyranoside showed maximum zone of inhibition 34 mm, 31 mm, 30 mm and 28 mm against E. coli, Salmonella, K. pneumonia and S. aureus respectively while less active against P. aeruginosa, S. epidermitis and Xanthomonas with inhibitory zones 8 mm,16 mm and 19 mm respectively.Mangiferrin was more active against K. pneumonia (32 mm) and Salmonella (28 mm) and weak against P. aeruginosa (10 mm), E. coli (15 mm).The compound isomangiferrin contained strong antibacterial activity against S. aureus (32 mm) and K. pneumonia (28 mm) while lowest against 5 mm against P. aeruginosa.Astragalin, tiliroside and quercetin-3-O rhamnoside) showed strongest activity against K. pneumonia with inhibitory zone i.e. 26 mm, 24 mm, and 22 mm respectively.Similarly strongest antibacterial activity of Irriflophenone-3-C-β-D-glucopyranoside, mangiferrin and isomangiferrin was observed 21,23 .The compound of R. brunonii i.e. astragalin, tiliroside, quercitin-3-Orhamnoside showed maximum inhibitory zone 26 mm, 24 mm, and 22 mm respectively against Klebsiella pneumoniae, while lowest against P. aeruginosa.Comparison of compounds antibacterial potential against different  www.nature.com/scientificreports/strains (Fig. 3).In previous study it has been observed that methanolic fruit's extract of R. Brunonii showed highest antibacterial potential against Klebsiella pneumoniae and lowest against Bacillus subtilis 18 .
For cytotoxic assessment of compounds brine shrimp lethality assay was used.The compound tiliroside exhibited 95% mortality rate with LD 50 value 73.59 µg/mL highest mortality rate as compared to all other compounds.Harmaline showed 80% cytotoxic effect while harmine and Irriflophenone-3-C-β-D-glucopyranoside showed 65% mortality at highest 200 µg/mL concentration.The 55% was observed by vasicine, isomangiferrin and astragalin at 200 µg/mL.The lowest 50% cytotoxicity was showed by compound mangiferrin whereas 60% cytotoxic effect was observed by quercetin-3-O rhamnoside at 200 µg/mL (Fig. 4).Nicotine was used as standard.It showed 100 percent mortality rate at 200 µg/mL with lowest LD 50 value 30.69 µg/mL among all compounds (Table 3).In previous literature cytotoxicity of mangiferrin and isomangiferrin has been observed 21 .Antibacterial, antioxidant and cytotoxic effects of some compounds were checked for the first time, but in previous literature same work has been done on whole plants and different fractions of these plants (P.harmala, D. ramosa and R. brunonii).These above different compounds were isolated from these plants.That's why compounds also exhibited such potentials like antibacterial, antioxidant and cytotoxic potentials.
In the process of drug discovery, computational innovations had a substantial impact.Computational biology emerged as an interdisciplinary approach integrating the computational techniques with biological systems to target different disease related challenges 30 .Virtual screening methods are frequently used to reduce the cost and time of medication development.Virtual screening predicts both binding modes and affinities of receptor and ligand based on docking and scoring of each molecule in a data set.Molecular docking has aided important drug discovery procedures.Docking approaches aid in the identification of correct ligand poses in a protein's binding pocket as well as the prediction of ligand-protein affinity.Finally, docking is a three-dimensional process for fitting two molecules together [31][32][33][34][35] .
In this study, molecular docking of different compounds that were previously reported from three medicinal plants was also done with target proteins (anticancer and antiviral).Similar study was reported by Gayathiri    www.nature.com/scientificreports/et al., 30 in Sauropus androgynus (L.) Merr against inflammation and cancer.The biochemical compounds were taken ligand to evaluate the antiviral and anticancer efficacy of these compounds.The 3D structures of antiviral and anticancer proteins were obtained from RCSB Protein Data Bank.Docking results were represented in the form of e-negative values (Tables 4 and 5).The arrangements of ligands (compounds) were based on ligand-protein binding energies.In molecular docking, higher e-negative values represent high binding affinity between the receptor and ligands molecules that indicated the higher efficacy of bioactive compound.

Conclusion
Plant derived bioactive compounds have several therapeutic effects.Present study is highlighting the importance of bioactive compounds of P. harmala, D. ramosa and R. brunonii with significant antibacterial, cytotoxic and antioxidant potential in vitro and in silico that justified the ethnomedicinal importance of these plants.The compound tiliroside (R. brunonii) showed highest (95%) cytotoxic effect on brine nauplii among all compounds.The current study also explored the interaction of bioactive compounds to inhibit different cancer (2XV7 & 5C5S) and viral (Hepatitis 6VAR, SARS Covid 19 6LUS & Influenza IEA3) proteins.The results from this study displayed that compound tiliroside demonstrated the high binding affinity towards all cancer and viral proteins as compared to all other compounds.These bioactive compounds are significant sources of natural antioxidants, which could help to prevent the progression of various diseases, caused by free radicals i.e. certain cancers.In conclusion these results suggest that bioactive compounds may be helpful for the development of newer and more effective pharmaceutical components which may have lesser adverse effects.

Antioxidant assessment
The antioxidant activities of compounds were analyzed by DPPH Free Radical Scavenging Assay and ABTS Assay.

DPPH (2-2-diphenyl-1-picrylhydrazyl) Free Radical Scavenging Assay
The DPPH assay was performed according to the protocol of Ghasemi et al. 40 with little modifications.For DPPH solution, 3 mg of DPPH powder was dissolved in 100 mL methanol.4 mg of compounds were dissolved in 5 mL methanol to obtained stock solution of compounds.Different serial dilutions (25, 50, 100, and 200 μg/mL) were prepared.The 3 mL DPPH methanolic solution was added to 400 μL compounds methanolic solution.The mixture was continuously shaken and stored at room temperature under shade for exactly 30 min.The absorbance of reaction mixture was measured at 515 nm spectrophotometrically.The dark violet color changed to light yellow color.The experiment was repeated three times and percentage inhibition was calculated as follows.
IC 50 value was calculated by linear regression equation that was obtained by plotting concentrations against percentage scavenging activity.

ABTS Assay (2,2 -azino-bis-3-ethyibenzothiazoline-6-sulfonic acid)
The ABTS test was performed using technique with some alternations 41 .The stock solutions (7 mM ABTS solution in 100 mL distilled water) and (2.4 mM potassium persulfate in 100 mL distilled water) were prepared.The ABTS solution was prepared by mixing equal parts of the two standard stock solutions and left them to react for 14 h at 25 °C in the dark.For compound solution, 4 mg of compounds were dissolved in 5 mL distilled water.Different concentrations (25, 50, 100 & 200 μg/mL) were prepared by using this stock solution.400 μL compounds solution was allowed to react with 3 mL of the ABTS solution and the absorbance was taken after 30 min.Ascorbic acid was used as positive control.Color was changed from bluish green to colorless.The percentage scavenging potential was calculated by using following formula.
Absorbance blank is absorbance of control (without compound) and Absorbance sample is absorbance of sample (compound).
IC 50 value was calculated by linear regression equation that was obtained by plotting concentrations against percentage scavenging activity.

Antibacterial activity
Antibacterial activity of compounds was determined by agar well diffusion method 42 .Antibacterial activity of different compounds was tested against seven clinical pathogens (Klebsiella pneumonia, Escherichia coli, Xanthomonas, S. aureus, Salmonella, P. aeruginosa and S. epidermitis).The nutrient agar medium was prepared by dissolving 6 g agar nutrient powder in 250 mL distilled water.The media was autoclaved and poured into www.nature.com/scientificreports/pre-labeled petri plates under sterile condition.Then rinsed these plates with bacterial suspension before solidification.Allowed it to solidify for 15 min.Compound solution of 0.6 mg/mL was used.Cefixime with 1 mg/1 mL was used as positive control whereas DMSO was used as negative control.After solidification made wells of 6 mm using sterile cork borer.Each well was sealed with 20 μL of agar.After sealing 40 μL of each compound solution was added in well.The petri plates were sealed by para-film and kept in incubator for 24 h at 37 °C.The experiment was run in triplicates.The zone of inhibition of bacterial growth was measured with scale in mm.

Cytotoxicity assessment
Brine shrimp lethality assay (BSLA) was used to check out the cytotoxic potential of different compounds of plant species by using the protocol described by Ishaque et al. 18 with several modifications.Artificial sea water was prepared by dissolving 38 g sea salt in 1000 mL distilled water.This sea water was poured in a two-chamber container.One chamber was big and other was small.Added brine shrimp's eggs on small side of chamber.Small side of chamber was covered and on other side provided illumination with electric lamp.Electric lamp was used to attract brine shrimp's larva photo-tactically.Kept this for 24 h at room temperature for hatching of eggs.After 24 h brine shrimp's larva matured to nauplii.Picked these nauplii from illuminated side with glass capillary tube for further process.The compounds were dissolved in Dimethyl sulfoxide (DMSO) and different concentrations of these compounds were prepared (25, 50, 100 and 200 μg/mL).Nicotine was used as positive control and same concentrations were made.Artificial sea water was used as a negative control.These concentrations were poured in vials and final volume of 5 mL was made by adding sea water.Added phototropic 20 nauplii in each vial with glass capillary tube.Then these vials were kept at room temperature for 24 h for incubation.After that alive nauplii were calculated.Mortality rate of brine shrimps was measured by using formula: LC 50 was calculated by drawing graph between percentage mortality and concentrations using excel.

In silico analysis
Plant extracts possess thousands of bioactive chemicals, and determining the biological efficacy of each one in laboratory is too time consuming 43 .Therefore in silico evaluation of bioactive compounds from medicinal plants targeting cancerous and viral proteins has been done.Following steps were involved in Insilico analysis.

Molecular docking studies
The "key and lock" theory is utilized in molecular docking to discover the best match orientation for ligand and protein 44 .The structures of compounds were obtained from Pub Chem in SDF format.The phytochemicals show potential as drug candidates against the selected proteins by satisfying the Lipinski rule.Which entails a molecular weight below 500 g/mol, fewer than 10 H-bond acceptors, and fewer than 5 H-bond donors.Molecular weight, indicative of density, size, volume, and mass, is a key factor for therapeutic agents 45 .Furthermore, adherence to other rules, such as a molar refractivity between 40 and 130 and having fewer than 10 rotatable bonds, additional supports the drug-likeness 46 .Lipinski's rule-of-five, which connects physicochemical and pharmacokinetic indices, underlines that an orally active drug-like compound should not defy more than one of the subsequent criteria: hydrogen bond donors not exceeding 5, hydrogen bond acceptors not exceeding 10, molecular weight not exceeding 500 Da, and octanol-water partition coefficient (log P) not exceeding 5. Importantly, all selected phytochemicals adhere to these criteria.Anticancer (2XV7 and 5C5S) and antiviral proteins (hepatitis 6VAR, influenza 1EA3, SARS Covid-19 6LUS) were obtained from protein database.The target protein and ligands were synthesized according to normal ligand and protein preparation procedures, and the protein and ligand files were sent to Auto Dock vina.Each ligand's binding energy and binding contacts were determined.The docked complexes were studied using Ligplus.

Ligand preparation
The 3D structures of compounds (ligands) were obtained from Pub Chem compound database at NCBI in SDF format (Table 6).Then changed these SDF format structures to PDB format using pymol.Ligands were prepared by removing water molecules, adding hydrogen bonds, and required charges (Kollman and Gasteiger) using AutodockTools-1.5.7.Then saved structures of compounds in PDBQT format and used for docking by using AutodockTools-1.5.7 47 .

Protein preparation
The crystal structures of different proteins were retrieved from RCSB Protein Data Bank in PDB format 48 .These proteins were anticancer (2XV7 & 5C5S) and antiviral (influenza 1EA3 & hepatitis 6VAR) (Fig. 5).These raw PDB proteins have only water molecules, metal ions, heavy atoms, and co-factors.These protein structures contained no information about formal atomic charges, bond orders and topologies so could not be used for molecular docking studies.So removed all water molecules, added polar hydrogen atoms and charges (Gasteiger and Kollman) in protein crystal structure using AutodockTools-1.5.7.Then optimized structure by assigning bond angles, bond orders and topology.Saved these structures in PDBQT format for further analysis.

The generation of grid box
Pre-calculated grid map is important in Auto Dock.Grid map helps in calculation of docking score.The docking evaluation results were analyzed by determining the amino acids in the active site.A three -dimensional grid box was created at maximum on proteins, to allow ligands to be docked on all areas of the receptor 49 .To generate % of death = Total nauplii−Alivenauplii/Total nauplii × 100 grid box, selected both proteins receptor (5c5s.pdbqt,6var.pdbqt,2xv7.pdbqt,1ea3 & 6lus.pdbqt) and saved ligands (compounds) in pdbqt format.Grid map was generated in such a way that both ligand and receptor fit into this three-dimensional matrix.Adjusted grid box size for X,Y,Z dimensions.The space was adjusted as 0.5 A that was nearly quarter of length for carbon single bonding atom.In the center of grid box ligand was present and was well-adjusted inside the active side of receptor proteins.Then exported the grid box dimension as grid.txt format.Then saved it in respective file for further processing.

Use of command prompt
Command Prompt is command line interpreter tool.The command is issued at the command prompt to examine the ligand-receptor binding affinity.www.nature.com/scientificreports/ The analysis of protein-ligand complex by Ligplus For assessing and characterization of interactions in our docked protein-ligand complex, LIGPLOT + version v.2.www.nature.com/scientificreports/

Figure 1 .
Figure 1.Comparison of antioxidant potential of bioactive compounds and ascorbic acid in DPPH assay.

Figure 2 .
Figure 2. Comparison of antioxidant potential of different bioactive compounds of selected plant species and ascorbic acid in ABTS assay.

Figure 3 .Figure 4 .
Figure 3.Comparison of antibacterial potential of bioactive compounds in agar well diffusion method.
Inhibition (%) = Absorbance of control − Absorbance of sample /Absorbance of control × 100.(%) Inhibition = (Absorbance of control − Absorbance of sample)/Absorbance of control × 100 Vol:.(1234567890)Scientific Reports | (2024) 14:3736 | https://doi.org/10.1038/s41598-024-54470-6 2.4 was used to visualize compounds with the best binding affinities.The hydrophobic and polar interactions between both the ligand and the target protein were visualized using this software (Figs. 6, 7, 8, 9, 10).The ligand.pdbqt and protein.pdbqtopened in pymol at same time.Then clicked on file and exported molecule, given name as complex, saved in pdb format in respective folder.Again clicked on file, exported image as PNG, saved and give any name.The image.oxps was converted online to jpg in respective folder.This method was used to show and generate interactions between compounds.

Table 1 .
Calculation of regression equation, regression co-efficient and IC 50 values of bioactive compounds and ascorbic acid in DPPH assay.

Table 2 .
Calculation of regression equation, regression co-efficient and IC 50 values of bioactive compounds and ascorbic acid in ABTS assay.

Table 3 .
Calculation of regression equation, regression co-efficient and LD 50 values of bioactive compounds and ascorbic acid in BSL assay.

Table 4 .
Binding score of compounds against anticancer proteins 2XV7 and 5C5S.

Table 5 .
Summary chemically synthesized medicines.Additionally, previous studies have demonstrated the effectiveness of these plants in vitro in a variety of ways, including anticancer, antioxidant, antibacterial, and antiviral properties.
of binding affinities of compounds against hepatitis B virus protein (6VAR), influenza virus protein (1EA3) and SARS covid-19 protein (6LUS).Vol.:(0123456789) Scientific Reports | (2024) 14:3736 | https://doi.org/10.1038/s41598-024-54470-6www.nature.com/scientificreports/ The command prompt was open on desktop.Then provide command as "cd space the open folder of compound and add the link (E:\important\Molecular Docking\complete\cancer 2XV7) then enter " and go to computer, open local disk C, open programme file, then open "The Scripps Research Institute" open vina, copy link and added this link (C:\Program Files\The Scripps Research Institute\Vina).After that insert \, click on double tab, space, -config, space, grid.txt,space, -log, space, log.txt and press the enter button.After that docking results of compounds were obtained after analyzing of bonding score.

Table 6 .
Two dimensional structures of compounds with their Pub Chem CID and Molecular Weight.