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ORIGINAL ARTICLE
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Controlling non small cell lung cancer progression by blocking focal adhesion kinase-c-Src active site with Rosmarinus officinalis L. phytocomponents: An in silico and in vitro study


1 Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India
2 Department of Biochemistry, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad, Gujarat, India

Date of Submission07-Aug-2020
Date of Decision28-Sep-2020
Date of Acceptance25-Dec-2020
Date of Web Publication24-Jul-2021

Correspondence Address:
Hyacinth N Highland,
Department of Zoology, Biomedical Technology and Human Genetics, School of Sciences, Gujarat University, Ahmedabad - 380 009, Gujarat
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1064_20

 > Abstract 


Background: Non small cell lung cancer (NSCLC) is a global, fatal oncological malady to which conventional and targeted therapies proved less effective with consequent side effects; hence, phytocomponents from herbal sources may provide potent alternative and should be tested for cancer intervention. Activation and overexpression of proto-oncogene tyrosine kinase Src (c-Src) and focal adhesion kinase (FAK) lead to cell proliferation and invasion. Hence, in the present investigation, in silico analysis was carried out to identify molecular intervention of phytocomponents in blocking the active site and thus inhibiting c-Src and FAK activation, which in turn could control progression of NSCLC.
Materials and Methods: In silico analysis was carried out using Molegro Virtual Docker, Molegro Molecular Viewer, and ClusPro server for ligand–protein and protein–protein interaction study. Phytochemical analysis and assay for antioxidant activity of hydroalcoholic extract of Rosmarinus officinalis L. were carried out using standard phytochemical tests, high-performance thin-layer chromatography, and 2, 2-diphenyl-1-picrylhydrazyl assay. Effectiveness of extract in arresting cell proliferation was confirmed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay on A549 cell line.
Results: In silico analysis indicated effective binding of rosmarinic acid to the active site of target proteins FAK and c-Src, blocking their activity. MTT assay revealed potent antiproliferative activity of hydroalcoholic extract which acted in dose-dependent manner. Phytochemical analysis confirmed that the extract was rich in phytocomponents and had antioxidant activity of 94.9%, which could therefore effectively eliminate free radicals and inhibit cell progression.
Conclusion: In silico and in vitro studies confirmed that phytocomponents present in hydroalcoholic extract of R. officinalis L. could effectively block the active site of target proteins and thus controlled cell proliferation on NSCLC cells, suggesting herb as an effective alternative medicine for the treatment of NSCLC.

Keywords: Antiproliferative, focal adhesion kinase, non small cell lung cancer, proto-oncogene tyrosine kinase, Rosmarinus officinalis L.



How to cite this URL:
Highland HN, Thakur MB, George LB. Controlling non small cell lung cancer progression by blocking focal adhesion kinase-c-Src active site with Rosmarinus officinalis L. phytocomponents: An in silico and in vitro study. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322268




 > Introduction Top


Lung cancer is one of the most common fatal forms of cancer worldwide. Non small cell lung cancer (NSCLC) is the main histological type which accounts for 85% of all lung cancer.[1] In India, it is the second most prevalent cancer in men and fourth in women, accounting for 6.9% of all new cancer cases, and contributes to 8.82% of cancer-related mortality in both the sexes.[2] Lung adenocarcinoma, a subtype of NSCLC, is the most aggressive and rapidly fatal tumor type with a very low 5-year survival rate.[3] Conventional and targeted therapies are available, but fail to improve treatment due to development of resistance; hence, there is a need for new treatment options using novel alternatives to control NSCLC.[4]

The central focus in lung cancer treatment is to destroy tumor cells in the presence of normal cells, without damaging the latter. To overcome this challenge, therapeutic drugs from natural resources are currently being tested.[5] Medicinal plants are the most common source of novel natural products that aim at treating specific disease without damaging normal cells. Many natural products have also been tested as viable alternatives to current cancer treatments, since they are proven to have fewer side effects, great efficacy, and are cost-effective.[6] In India, herbal medicines in the form of plant extract mixtures have been used traditionally for centuries in folk medicine to treat various health problems;[7],[8] however, systematic research to investigate the potency is now warranted.

The proto-oncogene tyrosine protein kinase Src (c-Src) and focal adhesion kinase (FAK) are intracellular (non-receptor) tyrosine kinases that physically and functionally interact to promote variety of cellular responses.[9] The phosphorylation of c-Src is closely associated with cancer progression and it also participates in many signaling pathways, which include the FAK pathway, the activation of which ultimately promotes cancer cell survival, proliferation, and invasion. FAK localizes at focal adhesion sites and plays a vital role in tumor survival and metastasis. It has been shown to be overexpressed in lung cancer which suggests that its expression precedes invasion and metastasis, necessary for tumor survival signaling.[10]

Rosmarinus officinalis L., an aromatic medicinal herb commonly known as “Rosemary,” belongs to the family Lamiaceae which originates from the Mediterranean region.[11] Various phytocomponents such as phenols, flavonoids, and di- and tri-terpenoids have been identified in its extracts.[12] R. officinalis L. plant extract as well as its essential oils contains many biologically active components, as reported by Hamidpour et al.,[13] which are known to have hepatoprotective, antioxidant, anticarcinogenic, anti-inflammatory, and antiproliferative activities.

Hence, the present study was aimed at inhibiting the proliferation of NSCLC cells by natural phytocomponents from a medicinal herb that would consequently be safe, effective, and easily available.


 > Materials and Methods Top


In silico study

In the present study, Molegro Virtual Docker (MVD) v 5.0 (www.molegro.com)[14] was selected for molecular docking; along with this, Molegro Molecular Viewer (MMV) was used for calculating docking score. Protein–protein docking was done using ClusPro 2.0 online server.[15],[16]

Protein and ligand preparation

Protein–ligand interaction studies were performed on the selected phytocomponents from R. officinalis L. having potential therapeutic roles against the target protein FAK and c-Src.

The crystal structures of FAK (PDB ID: 4NY0)[17] and c-Src (PDB ID: 1FMK)[18] were obtained from the RCSB (Research Collaboratory for Structural Bioinformatics) (http://www.pdb.org/)[19] protein data bank. The selection of these structures was made according to the maximum matched sequences among the protein chains provided in the RCSB database and prepared for molecular docking simulation in such a way that all heteroatoms (i.e., nonreceptor atoms such as water and ions) were removed. 3D structure of all the selected phytocomponents from R. officinalis L. having a potential therapeutic role [Figure 1], used for docking studies were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/)[20] in structure data format.
Figure 1: 3D structure of the selected phytocomponents from different culinary herbs (https://pubchem.ncbi.nlm.nih.gov/)

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Molecular docking studies

Molecular docking was employed to understand the interaction of the selected phytocomponents, against the target protein FAK and c-Src. The proteins FAK and c-Src were further interacted in protein–protein docking with the help of ClusPro 2.0 online server.

Plant collection and identification

R. officinalis L. plant was selected for the present study because of its efficacious medicinal properties against diseases. The leaves of R. officinalis L. herb were collected from a home garden at Ahmedabad, Gujarat. Fresh leaves were collected and washed thoroughly and dried in shade to prevent the possible degradation of bioactive components. The dried leaves were ground to a fine powder and stored in airtight containers for further use. The authentication of the plant was done by Dr. Hitesh A. Solanki, Professor (Taxonomy), Department of Botany, School of Sciences, Gujarat University.

The plant powder was first defatted using petroleum ether (60°C–80°C) till complete defatted material was obtained. 5 g of this material was extracted in methanol and water in the ratio of 70:30 in a Soxhlet apparatus for up to 8–10 cycles till the solvent in the siphon of the Soxhlet apparatus became colorless. The extract was then filtered and dried at 60°C in a hot air oven and collected after fully dry. The dried material was then stored at 4°C for further phytochemical studies.

Qualitative phytochemical screening tests

The hydroalcoholic extract prepared by Soxhlation was then analyzed for the presence of various phytocomponents such as phenols, flavonoids, alkaloids, saponins, phytosteroids, proteins and amino acids, carbohydrates, reducing sugar, tannins, and terpenoids, following the standard methods described by Harborne.[21]

High-performance thin-layer chromatography analysis

High-performance thin-layer chromatography (HPTLC) is a commonly used technique for quantitative assays and also for identification of unknown phytocomponents from medicinal plants.[22] HPTLC was performed using precoated silica gel 60 F254 TLC plate; 20 μl of sample (crude extract dissolved in methanol) was applied on this plate using Linomat V autosprayer. The plates were developed using a mobile phase containing toluene: acetone: formic acid in the ratio of 6:6:1. The development was carried out in 10 cm × 10 cm twin trough glass chamber equilibrated with the mobile phase for about 20 min at 25°C ± 2°C. 10 ml of mobile phase was used for the development in a glass chamber and allowed to migrate at a distance of 70 mm. After development, the plate was dried and scanned under 254 nm (deuterium lamp) and 366 nm (mercury lamp) using Camag TLC scanner operated by winCATS planer chromatography software version 1.4.6.8121. The images were taken at 254 nm and 366 nm wavelengths by ultraviolet (UV)/visible lamp, in an ultraviolet cabinet attached with smart digital photographic unit (Camera), and the Rf values obtained were tabularized.

Free radical scavenging activity using 2, 2-diphenyl-1-picrylhydrazyl

The antioxidant activity of hydroalcoholic plant extract was determined by the standard method of Blois.[23] 1 mg/ml of stock plant extract was prepared and about 10 ml of 0.1 mM of DPPH was prepared and kept in dark until use. 1 ml of DPPH was added in 3 ml of different concentrations (3.9, 7.8, 15.62, 31.25, 62.5, 125, and 250 μg/ml) of R. officinalis L. extract, shaken, and mixed properly. The mixture was kept in dark for 30 min at room temperature; absorbance was read at 517 nm using a UV spectrophotometer. As a reference, ascorbic acid was used, DPPH and methanol were used as blank, and only DPPH served as negative control. The IC50 was calculated based on the absorbance. The percentage antioxidant activity (%AA) was calculated using the following formula:



Cell culture

The NSCLC cell line A549 (lung adenocarcinoma) was procured from the National Center for Cell Science, Pune, Maharashtra, India. The cell line was maintained according to the standard protocol of Roy et al.[24] with some modifications in Dulbecco's modified Eagle's medium/F-12 Ham (1:1 mixture) and supplemented with fetal bovine serum and antibiotic/antimycotic solution in a humidified 5% CO2 atmosphere in a CO2 incubator at 37°C temperature.

Antiproliferative activity by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay

Hydroalcoholic extract of R. officinalis L. leaves was analyzed for its antiproliferative activity against A549 cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, according to the principal of Wilson.[25] 1 × 103 cells/ml were seeded in a 96-well plate for 24 h, and after incubation, the media was removed and the cells were treated with the crude hydroalcoholic extract in time and dose-dependent manner (3.9–250 μg/ml for 24, 48, and 72 h). After particular interval of time, 10 μl of MTT reagent was added to each well and wrapped with aluminum foil to avoid the exposure of light. The wrapped plate was then kept in 5% CO2 incubator at 37°C for 2–4 h. The cells were observed for needle-shaped, dark purple precipitate (formazan crystals) at periodic intervals under an inverted microscope. The absorbance was read at 570 nm with reference wavelength higher than 650 nm using a spectrophotometer.

The average 570 nm absorbance values of the control wells were subtracted from the average 570 nm absorbance values of the corresponding experimental wells. The absorbance of all the assay wells was again measured at a wavelength higher than 650 nm and those values were subtracted from the values obtained at 570 nm.

The percentage antiproliferative effect (% growth inhibition) was calculated using the following formula:




 > Results Top


Docking analysis

Protein–ligand docking, and protein–protein docking are very useful approach for determining the binding interaction of a target protein with single or multiple ligands. Using different in silico tools such as MVD, MMV, and ClusPro, the results obtained indicate favorable binding interaction between the target proteins FAK and c-Src with phytocomponents which can inhibit their activation by blocking the activation sites.

Molecular docking of focal adhesion kinase

The docking study of the selected phytocomponents shows effective binding interactions with the target protein FAK. The ligands show binding affinity which is representative of various types of interactions including hydrogen bond interactions and steric interactions; the responsible key amino acid residues were Arg 127, Phe 253, Glu 256, Lys 255, and Tyr 347. After confirmation of these key residues in all the phytocomponents, rosmarinic acid showed the highest docking score of −130.551 with FAK [Table 1]. The binding pose of FAK and rosmarinic acid [Figure 2] shows that there are six hydrogen bond interactions and the key amino acid residues were Trp 97, Arg 125, Arg 127, Leu 129, Tyr 251, Phe 253, Lys 255, Glu 256, Ile 274, and Tyr 347.
Table 1: Interaction profile of focal adhesion kinase and phytocomponents

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Figure 2: Binding pose of focal adhesion kinase + rosmarinic acid

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Molecular docking of c-Src

The docking analysis of c-Src was also done similar to that described earlier for FAK. Molecular docking of the selected phytocomponents with the protein c-Src showed effective binding interaction and the key amino acids were Gln 251, Gln 253, Leu 317, His 319, and Gln 324. These residues have shown effective binding with most of the phytocomponents. Rosmarinic acid gave the highest docking score of −146.056 with the target protein c-Src [Table 2] giving five hydrogen bond interactions and the key residues were Asn 135, Ser 248, Lys 249, Pro 250, Gln 251, Gln 253, Met 314, Lys 315, Leu 317, Val 323, and Leu 325 [Figure 3].
Table 2: Interaction profile of c-Src + phytocomponents

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Figure 3: Binding poses of c-Src-rosmarinic acid

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Protein–protein docking

Protein–protein interaction study of FAK and c-Src was done using ClusPro 2.0 online server. The binding interaction of FAK and c-Src gave 30 model structures according to the interaction energy. The model structure which shows the highest binding energy of −262.4 was selected for further docking studies [Figure 4]. The protein–protein interaction of FAK and c-Src formed the complex structure FAK-c-Src, which was then used for the docking analysis.
Figure 4: Binding pose of focal adhesion kinase-c-Src (Chain-A) complex

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Molecular docking of focal adhesion kinase-c-Src

The selected phytocomponents were docked against the FAK-c-Src complex structure; chain A of both the proteins was used for the docking studies. The binding interaction of phytocomponents and FAK-c-Src complex shows the effective binding having Pro 290 (A), Ser 281 (A), His 292 (A) from FAK and Asp 190 (A), Cys 238 (A), and Asp 192 (A) from c-Src as amino acid key residues. Among all the phytocomponents, rosmarinic acid has the most effective binding interaction and the binding energy is −145.33 [Table 3] and the amino acid key residues were Glu 277 (A), Ser 281(A), Glu 278 (A) and His 292 (A) from FAK and Arg 169 (A), Cys 238 (A), Phe 191 (A), Asp 190 (A), Gly 236 (A), Pro 529 (A), Tyr 202 (A), and Gln 528 (A) from c-Src [Figure 5].
Table 3: Interaction profile of FAK – c-Src complex+Phytocomponents

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Figure 5: Binding poses of focal adhesion kinase-c-Src complex + rosmarinic acid

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Qualitative phytochemical screening tests result

The hydroalcoholic extract of R. officinalis L. revealed the presence of alkaloids, saponins, phytosteroids, proteins and amino acids, tannins, triterpenoids, phenols, flavonoids, and carbohydrates [Table 4].
Table 4: Phytochemical screenings of hydroalcoholic extract of Rosmarinus officinalis L

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High-performance thin-layer chromatography result

Phytocomponents present in the hydroalcoholic extract of R. officinalis L. leaves were confirmed with the help of HPTLC. The developed TLC plate is shown in [Figure 6] scanned under UV-254 nm and UV-366 nm. The results show the presence of 13 spots in the densitometric analysis under UV-254 nm with major peaks at Rf max 0.96, 0.79, 0.72, 0.48, and 0.23 with a percentage area of 20.59, 16.54, 16.76, 7.69, and 7.41, respectively. Minor peaks were also observed as shown in [Figure 7] and [Table 5].
Table 5: Number of detected peaks, their corresponding Rf max values, height–area calculation results of the hydroalcoholic extract of Rosmarinus officinalis L. at 254 nm wavelength (deuterium lamp)

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Figure 6: Developed TLC plate scanned under 254 nm and 366 nm wavelength

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Figure 7: HPTLC chromatogram of hydroalcoholic extract of Rosmarinus officinalis L. leaves under UV-254 nm

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The densitometric analysis of the plant extract under UV-366 nm revealed 14 spots with major peaks at 0.66 and 0.47 Rf max with a percentage area of 25.58 and 22.66. [Figure 8] and [Table 6] show the number of major and minor peaks at 366 nm.
Figure 8: HPTLC chromatogram of hydroalcoholic extract of Rosmarinus officinalis L. leaves under UV-366 nm

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Table 6: Number of detected peaks, their corresponding Rf max values, height–area calculation results of the hydroalcoholic extract of Rosmarinus officinalis L. at 366 nm wavelength

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DPPH assay results

The crude hydroalcoholic extract of R. officinalis L. leaves exhibited 94.9% antioxidant activity at 250 μg/ml of concentration [Figure 9]. The activity was compared with ascorbic acid as reference. IC50 for R. officinalis L. extract calculated was 10.568 μg/ml as compared to that of ascorbic acid of 8.708 μg/ml.
Figure 9: Percent antioxidant activity of Rosmarinus officinalis L. extract (IC50 = 10.568 μg/ml)

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3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay

A549 cells were treated with hydroalcoholic extract of R. officinalis L. leaves at different concentrations for 24, 48, and 72 h. The percentage decrease in growth proliferation of A549 cells is depicted in [Figure 10]. The percentage antiproliferative activity of plant extract was calculated by plotting the graph of concentration against % growth inhibition. The results indicated that there was a significant decrease in growth proliferation in a dose- and time-dependent manner. As the dose increased, the growth proliferation was observed to decrease. IC50 value was calculated by AAT Bioquest online software, which was found to be 15.551 μg/ml, 6.153 μg/ml, and 26.164 μg/ml after 24, 48, and 72 h, respectively.
Figure 10: Graphical representation of percent growth inhibition of A549 cells after treatment of Rosmarinus officinalis L. extract for 24, 48, and 72 h

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 > Discussion Top


Despite the availability of modern therapies and medications, lung cancer is still the major cause of death in developed and developing countries.[26] Most lung cancer cases are managed by chemotherapy, but it is not without profound side effects. Researchers have confirmed that phytocomponents derived from medicinal herbs are effective, inexpensive, and nontoxic. The phytocomponents present in the plants make them an appropriate choice for herbal drugs.[27] These naturally occurring active components are now receiving attention for their medicinal properties in treating several diseases;[28] however, scientific validation for their therapeutic application is imperative. Hence, in the present study, phytocomponents present in R. officinalis L. extract were investigated in silico and in vitro for their efficacy in controlling cell proliferation.

According to Zhou et al.,[29] FAK acts as a signaling domain for multiple proteins participating in downstream signaling pathways and is responsible for various functions in cell migration and cell survival which ultimately results in cancer progression. FAK comprises two noncatalytic domains such as FERM and FAT. The activation of FAK requires autophosphorylation which offers a binding site for c-Src. Binding of c-Src to FAK triggers the phosphorylation of the domains which thereby increase the FAK activity and activation of c-Src, leading to enhanced invasion, cell migration, and metastasis.

In the present study, the in silico analysis showed strong binding affinity of various phytocomponents from R. officinalis L. with evidence that the binding scores of rosmarinic acid with FAK and c-Src, i.e., −130.551 (Kcal/mol) and − 146.056 (Kcal/mol), respectively, were the lowest, indicative of strong, effective binding. These results validate the binding potential of rosmarinic acid with the active site of the target proteins, suggesting that this herbal constituent could effectively block FAK activity. Further, these results were supported by in vitro analysis.

Jafari et al.[30] observed that phenolic components present in plant extracts have direct control on cell cycle and flavonoids can also modulate many biological events in cancer such as cell differentiation and progression and can inhibit cell proliferation in various types of cancer. In a separate study, Batra and Sharma[31] have indicated that flavonoids also influence the immunological events which are associated with progression of cancer cells and hence cell proliferation. In the present study, the HPTLC analysis of the plant extract revealed the presence of various phytocomponents including phenols and flavonoids in abundance which evinced that it may consequently influence certain pathways within the A549 cells and inhibiting the growth of transformed cells. In addition, these molecules may play a role in the binding interaction of the components of R. officinalis L. and target proteins FAK and c-Src.

Natural antioxidants also play a central role in the prevention and treatment of many diseases. Andrade et al.[32] have reported that reactive oxygen species including hydrogen peroxide and free radicals are inevitably produced in living organisms which result from metabolic processes. In our study, the plant extract tested showed highly significant free radical scavenging activity of 94.9%, which would eliminate the free radicals which impede various biochemical and signaling reactions, resulting in uncontrolled cell proliferation.

The antiproliferative effect of the hydroalcoholic extract of R. officinalis L. leaves indicated the inhibition of cell growth in a dose- and time-dependent manner in the range of 37.3%–89.66% when treated for 24 h, 48 h, and 72 h. In a separate investigation on colon cancer cell line, Slamenova et al.[33] have demonstrated that R. officinalis L. extract decreases cell proliferation with IC50 value of 30 μg/ml (24 h) on colon cancer cell line. Similarly, Yi and Wetzstein[34] have examined the antitumorigenic activity of this plant extract with IC50 value of 71.8 μg/ml. These observations lend support to our results of the antiproliferative action of R. officinalis L. extract on the NSCLC cell line. Hence, the present investigation revealed that R. officinalis L. is effective on A549 cell line in a dose-dependent manner. The results in the present study affirm that at a specific concentration, the extract can inhibit cell proliferation possibly by blocking the active sites of the target proteins FAK and c-Src which would inhibit the binding of proteins responsible for downstream signaling pathways and can reduce the uncontrolled cell proliferation.


 > Conclusion Top


The study examined the effect of various phytocomponents with the help of in silico tools which revealed that rosmarinic acid manifested the most potent binding interaction with the target proteins FAK and c-Src, could therefore block their binding sites, and suppress the overexpression of these proteins in NSCLC. The decrease in the cell proliferation of A549 cells demonstrated by the MTT assay confirmed the effectiveness of R. officinalis L. hydroalcoholic leaf extract. Phytochemical analysis confirmed the presence of phenolics and flavonoids which could contribute to the effectiveness of this plant extract. In addition, the extract was also found to have potent antioxidant activity.

Hence, phytocomponents of R. officinalis L. could yield lead molecules in the effective control of NSCLC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

 
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