Docking tools provide perfect leads for Cytotoxic activity of Lupenone and Sitostanol caffeate from root extracts of Roscoea purpurea

Gunpreet Kaur¹, Ravinder Sharma², Parveen Bansal¹, Ramandeep Kaur¹, Vikas Gupta¹*

¹University Center of Excellence in Research, Baba Farid University of Health Sciences, Faridkot, Punjab, India.

²Faculty of Pharmaceutical Sciences, ICFAI University, Baddi, India.

*Corresponding Author E-mail: vikas_4308@rediffmail.com

ABSTRACT:

Cancer is one of the most prevalent diseases and is increasing progressively due to our contemporary lifestyle. It is essential to find novel approaches to prevent and treat it efficiently. In order to treat cancer, herbal medications play a crucial role by obstructing important biological pathways. The current investigation was conducted to find out cytotoxic activity of four isolated compounds from root extracts of Roscoea purpurea. The isolated compounds were screened for cytotoxic activity through molecular docking studies using protein target 6G9X and 4DDR followed by in vitro cytotoxic using lymphoma cell lines i.e. Jurkat and u937. The molecular docking results showed that Lupenone and Sitostanol caffeate have cytotoxic activity. Lupenone and Sitostanol caffeate showed the higher/similar selectivity score as compared to standard drug Methotrexate. In consonance to the results of molecular docking, appreciable in vitro cytotoxic activity of Lupenone and Sitostanol caffeate has been observed through MTT assay. From results, it was observed that both the compounds showed decreased cell viability and inhibition of cell growth in a dose dependent manner. However more studies are needed to determine the exact mechanism by which these phytochemical constituents isolated from Roscoea purpurea interact with receptors to exert the cytotoxic activity.

KEYWORDS: Roscoea purpurea, Cytotoxic activity, Molecular docking, Lupenone, Sitostanol caffeate.

INTRODUCTION:

Cancer is a dynamic process which may be described as an uncontrolled tissue growth that results from an unbalanced relationship among cell division and cell death¹. Despite tremendous advancements in the development of anticancer therapies, the prevalence of cancer is still rising globally. According to the International Agency for Research on Cancer (IACR), there were around 10 million deaths due to malignancy and about 19.3 million new instances of cancer worldwide in 2020². Similarly, India has 1.32 million new disease cases (0.64 million men and 0.67 million ladies) and 0.85 million cancer deaths in 2020³.

Chemotherapy, radiation and surgery are some of the current therapeutic modalities having a limited involvement in disease eradication. Though multidisciplinary scientific approaches and research are making possible attempt to combat this disease, a satisfactory treatment has not yet been introduced into global health to the benefit of humanity. Keeping in view the plethora of adverse effects associated with the currently available pharmacological molecules, cancer patients are now turning towards complementary and alternative medicine for support^2-3.

In terms of chemoprevention, herbal drugs and dietary phytochemicals have been positioned at the top of the pyramid^4-5. Research studies demonstrate that phytochemicals can alter the complex multistage complicated process of carcinogenesis⁶. About 35,000 medicinal plant species have been identified that are used in the treatment of cancer however National Cancer Institute (NCI) has screened 3000 plants as potent anticancer agents⁷. According to a survey, it was found that 90 of the 121 prescription medicines used in treatment of cancer came from plant species and 74% of these medicines were discovered by conducting research studies on a folklore claim⁸. Many of these natural dietary phytochemicals are on the cusp of completing phase III clinical trials, regardless of the fact that the chemo-preventive effect of these compounds was initially predicted on cell line culture, in-vitro and animal model research studies⁹. In order to lower the steadily rising incidence of cancer, chemoprevention using naturally existing food components is seen as a realistic strategy. In continuation to this drug development, a number of phytochemicals have been screened and various single molecules have been isolated. The bioactivity guided fractionation is a very tedious and time consuming process. Hence to reduce this time frame, molecular docking studies combined with cell line studies can give a preliminary picture of cytotoxic activity of any compound and can be of tremendous use^10-12.

The incredible plant Roscoea purpurea (also known as kakoli), which has been utilised by saints and rishies for centuries, is now entered in the list of plants species that are in danger of going extinct for a variety of causes. Ancient literature suggests that formulations including kakoli have extremely effective uses, but the knowledge is only available in regional (local) languages, thus, the scientific community is not well-versed in the plant's actual use. Tubers of Roscoea are recognized as healthy aphrodisiac tonic that nourishes body and functions as an antioxidant in the body¹³. It is an established fact that most of the antioxidants have been found to be having anticancer activity also. Hence anticipating the anticancer activity with the accomplished preliminary work on isolation of four compounds named Lupenone, Epigallocatechin, Epicatechin and Sitostanol caffeate respectively from root extracts of Roscoea purpurea^14-16. Instead of opting bioactivity guided fractionation, it was decided to apply molecular docking tools to ascertain the anticancer potential of these isolates. Hence, In present study, authors made an effort to explore the cytotoxic potential of isolated compounds from Roscoea purpurea using molecular docking against target 6G9X and 4DDR by employing AutoDock-Vina¹⁷ and in-vitro cytotoxic activity using standard drug Methotrexate on human lymphoma cell lines.

MATERIALS AND METHODS:

Isolation of four compounds named Lupenone, Epigallocatechin, Epicatechin and Sitostanol caffeate was done using column chromatography and characterization of compounds was done using IR, NMR, and mass spectroscopy techniques from root extracts of Roscoea purpurea^14-16. Lymphoma cell lines Jurkat and u937 used for the cytotoxic study were procured from NCCS (National Centre for Cell Science), Pune.

In-silico activity:

The X-ray crystallographic structures of various protein targets 6G9X and 4DDR were downloaded from RCSB-PDB (Research Collaboratory for Structural Bioinformatics-Protein Data Bank)¹⁷. The protein targets 6G9X and 4DDR, which have shown the best X-ray crystallographic structures at resolution 2.30Ǻ and 2.05A respectively, were used as the receptor in the molecular docking experiments. After retrieving the structure from the RCSB-PDB, all the water molecules as well as hetro atoms were discarded and polar hydrogen atoms were added to the protein by using Biovia discovery studio visualizer (BDSV)¹⁸. The various active sites were determined using the site sphere method, and energy minimization was done using the MMFF94 force field. After that, the protein targets were ready for docking. The PROCHECK SAVESv6.0 server was widely used to check the integrity of generated 3D crystallographic and NMR structure model of various proteins 6G9X and 4DDR by uploading the (.pdb) file of the prepared proteins^19,20. The stereochemical quality and accuracy of the selected models were evaluated with a PROCHECK generated Ramachandran plot which depicts the statistical distribution of the arrangement of the backbone dihedral angles ϕ (phi) and ψ (psi) which can be used for structure validation^21-22.

The 3D structure of Citrus paradisi phytochemicals were downloaded from PubChem and ChemDraw-3D v19 in .sdf and .mol2 format which was further converted into .pdb format by using software Open Bable GUI²³. The ligands were then activated for docking by energy minimization through MMFF94s force field. Molecular docking is considered as the most admired virtual screening techniques, particularly when the target protein's 3D structure is available as it identifies the protein-ligand complex structure and the binding affinity between them, which is crucial knowledge for lead optimization. Once the protein targets and ligands were prepared, molecular docking was done with the aid of software autodock-vina embedded in PyRx²⁴. After completion of docking, the best optimal conformations were further analysed for binding energy (Kcal/mol) with the help of docking score²⁵. The 2D and 3D best binding structures thus obtained with the lowest energy were selected for further analysis using BDSV.

In- vitro cytotoxic activity:

Compound showed good docking score as compared to standard drug were selected and stock solution was prepared by dissolving the respective compounds (1mg/ml) in DMSO. Four different working concentrations (5, 10, 20, 40 and 80µg/ml) were then prepared from stock solutions. Prior to use, all solutions were sterilized by passing through 0.22μm syringe-adapted filters. Cytotoxicity of the isolated compounds was evaluated on human lymphoma cell lines i.e. Jurkat (P. No. 35) and u937 (P. No-40). The cells were allowed to grow in RPMI-1640 medium (L-glutamine), supplemented with hypoxanthine (10μg/ml), NaHCO3 (2mg/ml), Fetal Bovine Serum (10%), glucose (11.1 mM), and antibiotic solution (5μg/ml). The cells were then incubated at 5% CO₂ in humidified incubator at temperature 37°C until confluent stage. Following incubation, as the suspended cells attain confluency stage, 1/3^rd of spent medium was aseptically removed and replaced with the equal amount of fresh medium. Healthy lymphoma cells of the cell lines i.e. Jurkat and u937 were then transferred at concentration of 10,000 cells/100 μl/well to 96 well plates and incubated at 37°C for 48 h before addition of the compounds. Furthermore, 100µl of different working concentrations (5, 10, 20, 40 and 80µg/ml) of compounds were then added to wells of 96 well plate. The wells containing only cell suspensions without active compound were nominated as controls. The methotrexate (anticancer drug) was used as the standard drug and was tested in triplicate. The plate containing cells with or without active compounds and the standard drug were incubated for 72h at 37°C before determining cell viability, turbidity and toxicity^26-28.

MTT assay was done for determining cell viability using standard method²⁹. The OD (optical density) of plate was then measured using DMSO as blankat 540nm and % cell viability was calculated using the following formula: Cell viability % = Mean OD of wells receiving each plant extract concentration/ Mean OD of control wells x 100.

RESULTS AND DISCUSSION:

In-silico activity:

The 3D X-ray crystallographic and NMR structure of both proteins i.e. 6G9X and 4DDR was validated by the PROCHECK web server, which generates Ramachandran plot. The Ramachandran plot of the protein 6G9X (Figure 1) represents 97.3% of total residues in the most favored region, 2.7% in the additionally allowed region and about 0.0% in the generously allowed region. Similarly the protein 4DDR represents 90.6% of total residues in the most favored region, 9.4% in the additionally allowed region and 0.0% in the generously allowed region is given in Figure 2, indicating good quality models for study. Molecular docking generates several potentials adduct structures which are ranked and categorized by using the scoring function in the software programme. The docking outcomes showed that 2 phytochemicals named Lupenone and Sitostanol caffeate out of 4 isolated compounds exhibited good binding affinity with target molecules (6G9X and 4DDR) as compared to standard drug (Methotrexate). The binding affinity of various active phytochemicals along with reference drugs has been given in Table 1. Lupenone and Sitostanol caffeate which shows higher binding energy were further screened in-vitro for their probable cytotoxic activity against human cell lines Jurkat and u937.

Figure 1: Ramachandran plot of a protein 6G9X

Figure 2: Ramachandran plot of a protein 4DDR

Table 1: The binding energy of isolated compounds from roots extracts of Roscoea purpurea against therapeutic targets for cytotoxic activity

Sr. No	Phytochemicals and CID’s	SMILES	Binding energy of 6G9X	Binding energy of 4DDR
1.	Lupenone CID: 92158	CC(=C)C1CCC2(C1C3CCC4C5(CCC(=O)C(C5CCC4(C3(CC2)C)C)(C)C)C)C	-8.4	-9.7
2.	Epigallocatechin CID: 72277	C1C(C(OC2=CC(=CC(=C21)O)O)C3=CC(=C(C(=C3)O)O)O)O	-6.8	-8.8
3.	Epicatechin CID:72276	C1C(C(OC2=CC(=CC(=C21)O)O)C3=CC(=C(C=C3)O)O)O	-6.7	-8.6
4.	Sitostanol caffeate CID:222284	CCC(CCC(C)C1CCC2C1(CCC3C2CC=C4C3(CCC(C4)O)C)C)C(C)C	-8.0	-10.5
5.	Standard drug: Methotrexate CID: 126941	CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)NC(CCC(=O)O)C(=O)O	-8.0	-9.7

Table 2: The 3D interactions of 6G9X and 4DDR proteins with different ligands.

S. No.	Isolated Compounds (Ligands)	6G9X	Bonds	4DDR	Bonds
1.	Lupenone CID: 92158		Pi alkyl bond-Phe A:65,Leu A:357		Conventional Hydrogen bond-Lys A:55 Pi alkyl bond-Phe A:34,Leu A:22
2.	Sitostanol caffeate CID:222284		Pi-sigma bond-Phe B:254 Pi alkyl bond-Phe B:311,Bng A:509,Ala B:258,Phe B:254		Pi-sigma bond-Phe A:34,Phe A:31 Pi alkyl bond- Ala A:9,Ile A:16,Tyr A:121,Leu A:22,Pro A:26,Pro A:61,Phe A:31
3.	Standard drug: Methotrexate CID: 126941		Conventional Hydrogen bond-Gly B:381,Asp B:257 Pi alkyl bond-Ala B:258		Conventional Hydrogen bond-Leu A:27,Glu A:30,Gly A:20, Asp A:21, Ser A:118 Carbon Hydrogen bond-Ile A:7 Pi alkyl bond- Ile A:16,LeuA:22 Pi-pi stacked bond-Phe A:34 Pi-sigma bond-Leu A:22

In-vitro cytotoxic activity:

After taking leads from molecular docking studies, Lupenone and Sitostanol caffeate were screened for cytotoxic activity against Jurkat and u937 cell lines. The results showed decreased cell viability and cell growth inhibition in a dose dependent manner. The IC₅₀ of Lupenone and Sitostanol caffeate and methotrexate (standard) were 31.24μg/ml, 38.44μg/ml and 22.29μg/ml respectively for Jurkat and 32.38μg/ml, 38.24μg/ml and 16.73μg/ml respectively for u937 (Figure 3 and Figure 4.). Lupenone demonstrated strong anti-proliferative activities. According to accumulating data, apoptosis is a crucial biological target for dietary bioactive substances in the cancer prevention. The different extracts of P. microphyllum and P. mucronatum has been shown to exert cytotoxic activity against different cell lines due to the wide variety of constituents like lupenone, lupeol, hexacosane etc³¹.

Figure 3: In-vitro cytotoxic activity of Lupenone and Sitostanol caffeate on Jurkat cell lines

Figure 4: In-vitro cytotoxic activity of Lupenone and Sitostanol caffeate on u937 cell lines

CONCLUSION:

The current study showed that Lupenone and Sitostanol caffeate isolated from root extracts of Roscoea purpurea showed significant in-silico and in-vitro cytotoxic activity as compared to standard drugs. Lupenone could be a novel source of development of new plant based anticancer drug. This study also recommends use of molecular docking studies as a tool using suitable ligands for initial screening of compounds for anticancer activity to avoid loss of time in drug development. Further research is to be carried out to determine the exact mechanism for cytotoxic activity.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Baba Farid University of Health Sciences, Faridkot for their kind support.

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Received on 02.08.2023 Modified on 17.01.2024

Research J. Pharm. and Tech 2024; 17(7):3067-3072.

DOI: 10.52711/0974-360X.2024.00480