In silico molecular docking and in vitro analysis of ethanolic extract Ocimum sanctum Linn.: Inhibitory and apoptotic effects against non-small cell lung cancer

Abstract Background and Aim: Lung cancer, especially non-small cell lung cancer (NSCLC), has been identified as the leading cause of cancer deaths worldwide. The mortality rate from lung cancer has been estimated to be 18.4%. Until now, conventional treatments have not yielded optimal results, thus necessitating an investigation into the use of traditional herbal plants as potential candidates for its treatment. This study aimed to determine the inhibitory and apoptotic activity of the ethanolic extract from Ocimum sanctum Linn. (EEOS) by in silico molecular docking and through in vitro studies using NSCLC cells (A549 cell line). Materials and Methods: Dried simplicia of Ocimum sanctum was converted into EEOS using the maceration method. Spectrophotometry was then employed to analyze the EEOS compound. The known main active compounds were further analyzed for inhibitory and apoptotic effects on gene signaling using in silico molecular docking involving the downloading of active compounds from PubChem and target proteins from the Protein Data Bank; the active compounds and proteins were then prepared using the Discovery Studio software v. 19.0.0 and the PyRX 0.8 program, interacted with the HEX 8.0.0 program, and visualized with the Discovery Studio Visualizer v. 19.0. Finally, an in vitro analysis was performed using an antiproliferative-cytotoxic test (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay in the NSCLC A549 cell line). Results: The analysis revealed that the active compounds in the ethanolic extract were dominated by quercetin (flavonoids) (47.23% b/b) and eugenol (phenolic) (12.14% b/b). These active compounds interacted with the active sites (residual amino acids) of the αvβ3 integrin, a5b1 integrin, caspase-3, caspase-9, and vascular endothelial growth factor. Hydrogen bonds and Pi-cation and Pi-alkyl interactions were involved in the relationships between the active compounds and the active sites and thus may reveal an antioxidant property of the extract. Furthermore, in vitro analysis showed the inhibitory and antiproliferative effects of the EEOS against non-small cell cancer (A549). Conclusion: Taken together, our data showed the ability of EEOS as an inhibitor and apoptotic agent for lung cancer; however, further research is needed to determine the exact mechanism of EEOS as an herbal medication.


Introduction
Non-small cell lung cancer (NSCLC), a type of lung cancer, has been determined to be a significant cause of cancer death worldwide. Data from the Global Cancer Statistics show that the mortality rate due to lung cancer was 18.4% in 2018, with 1.8 million deaths and 2.1 million new lung cancer cases recorded [1]. Lung cancer is deemed the most fatal compared with other types of cancer. Although lung cancer is strongly correlated with smoking, adenocarcinoma of NSCLC can also occur in non-smokers, thus increasing the prevalence of lung cancer in all populations.
At present, most lung cancer treatments involve conventional therapy approaches (chemotherapy/ medication and surgery). Standard medication therapy in the treatment of NSCLC cases is the use of cisplatin in combination with pemetrexed [2]. In addition, treatment with antibody engineering systems such as pembrolizumab, nivolumab, and atezolizumab is now actively used [3,4]; however, the administration of these drugs has been noted to have drawbacks. Available at www.veterinaryworld.org/Vol.14/December-2021/ 16.pdf Aside from being expensive, these drugs induce a chemoresistant effect that appears in the treatment of lung and breast cancer. In addition, pembrolizumab and nivolumab are known to cause oral mucositis, rush, and pruritus caused by immune-related adverse effects [3,5]. Combination therapy such as radiotherapy with pembrolizumab is known to have side effects that increase the drug's toxic effect [5].
Because of the disadvantages of these medications, there is a need to explore natural herbal traditional remedies. The system of traditional medicine has been used for thousands of years to prevent, diagnose, and treat several acute and chronic diseases. Ocimum sanctum Linn. is a traditional medicine commonly found in Indonesia and Asian countries. Various species of Ocimum are known to provide many health benefits, including anti-inflammatory, anti-fatigue, antitussive, antiseptic, antispasmodic, neuroprotective, and neuroproliferative activities [6][7][8][9][10][11], but until recently, their active mechanisms, safety, and dosage have not been determined.
Thus, in this study, we aimed to determine the dynamic majority content of the ethanolic extract from O. sanctum Linn. (EEOS) and analyze the extract's effect against NSCLC by in silico molecular docking and in vitro studies.

Ethical approval
The study was approved by the Ethics Committee of the Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia (00053/EC/ FKH/Int./2021).

Study period and location
The study was conducted from January to

Flavonoid
EEOS, weighing 50 mg, was placed into a 10 mL test tube, in which 0.3 mL of 5% sodium nitrite (Merck) was subsequently added. After 5 min, 0.6 mL of 10% aluminum chloride (Merck) and 2 mL of 1 M sodium hydroxide (Merck) were added to the solution along with the addition of up to 10 mL of distilled water. The mixture was then transferred into a cuvette and measured through spectrophotometry (Shimadzu, Kyoto, Japan) at a wavelength of 510 nm. Quantification of the total compound was calculated using the following formula: The EEOS weighed 50 mg. To the extract, 0.5 mL of Folin-Ciocalteu (Merck) reagent and 7.5 mL of aquabides were added. The mixture was allowed to stand for 10 min at 24°C followed by the addition of 1.5 mL of 20% sodium carbonate (Merck). Sterile water was then added to achieve a final volume of 10 mL. The solution mixture was transferred into a cuvette and measured on a spectrophotometer (Shimadzu) at a wavelength of 760 nm. Quantification of the total compound was calculated using the following formula:

Cell proliferation assay (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide [MTT] assay)
The inhibitory activity of the EEOS was examined using an MTT assay. Cells that reached an 80% confluence in culture were detached with 1 mL of Accutase cell detachment (Capricorn Scientific). The cells were then inserted into a 15 mL sterile conical tube and centrifuged at 1500 rpm for 5 min. The supernatants were removed, and the cells were counted using a cell counter (Corning, New York, USA). Then, NSCLC (A549) cells were seeded onto a 96-well plate at a density of 1.5×10 4 cells/ well and incubated overnight at 37°C. Afterward, the cells were cultured with the AP3 monoclonal antibody at a concentration of 80 µg/mL as the antiproliferative positive control. In addition, 9 µg/mL cisplatin was run as the positive control of a commercial drug used to treat lung cancer. Furthermore, the EEOS was added in increasing concentrations (50, 70, 100, and 200 µg/mL). The cells were incubated for 24 h, washed with Dulbecco's phosphate buffer saline (Capricorn Scientific) followed by 10 µL of 5 µg/mL MTT (Merck), and incubated in the CO 2 incubator (Eppendorf, Hamburg, Germany) for 4 h at 37°C and 5% CO 2 . Finally, the medium was removed, and 100 µL of 95% sodium dodecyl sulfate 10% (Merck) was added to dissolve the formazan crystals. Optical density values were obtained using a microplate reader (Bio-Rad, California, USA) at 595 nm. Inhibition of the A549 cell line samples was calculated using the following formula: The NSCLC (A549) cells were seeded onto 6-well plates (at 5×10 5 cells/mL) for 24 h and then treated with 80 µg/mL of the AP3 monoclonal antibody inhibitor as the positive control, followed by 9 µg/mL of cisplatin as the commercial drug positive control, and the EEOS at increasing concentrations of 50, 70, 100, and 200 µg/mL for 24 h. The morphology of the cells was observed and photographed using a bright-field microscope (Nikon Eclipse TE2000-E, Tokyo, Japan). The observation data were analyzed semi-quantitatively.

Statistical analysis
The data obtained were analyzed using a oneway analysis of variance followed by a post hoc test by GraphPad Prism software v. 7 (La Jolla, CA, USA).

Eugenol and quercetin are the dominant components in the EEOS
The experimental analysis using an ultraviolet-visible spectrophotometer showed that EEOS consisted mostly of the active compound of flavonoid derivatives, that is, quercetin (47.23% b/b), and phenol derivatives, that is, eugenol (12.14% b/b) ( Table-1).

Quercetin binds to the active site of VEGF
Quercetin interacted with the amino acid residues in VEGF, namely, Gln79, Pro49, and Lys48,   Available at www.veterinaryworld.org/Vol.14/December-2021/16.pdf   Table-2). Hydrogen bonds and Pi-cation, P-sigma, and Pi-alkyl interactions were the foundation of the chemical interactions between quercetin and VEGF (Table-2).

Eugenol binds to the active sites of integrins αvβ3 and α5β1
Three amino acid residues were visualized from the interaction between eugenol and the αvβ3 integrin, namely, Ser342, Tyr406, and Arg261; these bonds produced affinity energy of 181.6 kJ/mol ( Figure-6 and Table-3). Meanwhile, the active compound in eugenol only bound to α5β1's amino acid residue, Leu257, yielded energy of −169.2 kJ/mol ( Figure-7 and Table-3). Hydrogen bonds and Pi-cation interactions established the chemical interactions between eugenol and integrins αvβ3 and α5β1 (Table-3).

Chemical interactions occurred between eugenol and caspase-3/caspase-9
The interaction of the eugenol ligand and protein caspase-3 produced one amino acid residue (Arg241) that bound the eugenol ligand (Figure-8), yielded energy of    (Table-3). Furthermore, the binding of eugenol and caspase-9 involved two amino acid residues that bound to eugenol on the active site of caspase-9, namely, Glu259 and Leu240 (Figure-9). The interaction of eugenol and caspase-9 resulted in binding energy of approximately −90.9 kJ/mol (Table-3). The interaction between eugenol and caspase-3/caspase-9 was established through hydrogen bonds and Pi-alkyl interactions (Table-3).

Eugenol bound to the active site of VEGF
The interaction of the eugenol compounds and the VEGF protein indicated the involvement of three amino acid residues (Leu97, Glu38, and Asn75) that interacted with the eugenol compounds through Pi-alkyl and hydrophobic interactions ( Figure-10 and Table-3). This interaction yielded a bond energy of −162 kJ/mol (Table-3).

Decreasing the viability of A549 cells in the presence of EEOS
NSCLC cells were cultured to evaluate the ability of EEOS to inhibit the cells' proliferation and adhesion to the extracellular matrix. Our results showed that EEOS significantly exhibited a cytotoxic Table-3: Interaction, chemical bond, and binding energy between eugenol and integrins αvβ3, integrins α5β1, caspase-3, caspase-9, and VEGF. c b a effect in human A549 cells, demonstrated by the percent-mean viability decrement in a concentration-dependent manner similar to cisplatin, and compared with the untreated control. The optimal concentration of EEOS was 200 µg/mL; at this EEOS concentration, there was a smaller number of viable A549 cells than the number seen with other concentrations of EEOS (50, 70, and 100 µg/mL) (Figures-11 and 12).

Discussion
Lung cancer remains the leading cause of cancer death in men and women worldwide [12]. In the past few years, the use of herbal medicine has continued to increase; currently, it is a trend to use herbal medicines concurrently with traditionally established treatments for cancer. One of the most   ; however, not much has been known about its use in the treatment of lung cancer. In this current study, we used in silico molecular docking and in vitro approaches to determine the mechanism of action of the EEOS.
Spectrophotometry analysis showed that EEOS contained two primary compounds, that is, quercetin and eugenol, belonging to the flavonoid group and the phenol group, respectively (Table-1). Some research has shown that natural sources (eugenol, caffeic acid, gallic acid, apigenin, quercetin, and rosmarinic acid) have therapeutic benefits in treating various diseases, including cancer [13][14][15][16][17]. In silico molecular docking, analysis showed that quercetin and eugenol were able to bind to the active site of the avb3 integrin (Arg99, Arg261, Tyr224, Lys409, Ser342, Tyr406, and Arg261) and a5b1 integrin (Thr258, Gly255, Asn256, Leu257, Ser277, and Leu257). Integrins are known to play a crucial role in mediating the adhesion of epithelial  cells to basement membranes. They also contribute to the migration, proliferation, and survival of tumor cells [18]. Furthermore, tumor cell expression of the integrins avβ3, a5β1, a6β4, a4β1, and vβ6 correlates with disease progression in various tumor types and is associated with cancer [18][19][20]. The adhesion of integrins to the extracellular matrix (ECM) provides the necessary traction for tumor cells' invasion. Integrins contribute to tumor cells' invasion by regulating the localization and activity of matrix-degrading proteases, such as matrix metalloprotease 2 (MMP2) and urokinase-type plasminogen activator (uPA). Integrinmediated migration generally requires focal adhesion kinase and Src family kinase signaling [18,21,22]. Cancer treatment could be promoted by inhibiting the activity of the integrin complex [22,23]. In addition, integrins physically associate laterally with cell membrane proteins (e.g., CD151 or CD47) to elicit or modulate signaling [24,25]. Data from this study indicated the potential for inhibition by the ligand on the integrin avb3 and integrin a5β1 proteins. Inhibition of the integrin complex would impact the inhibition of the extracellular matrix adhesion (ECM) process and limit tumor cells' invasion. In addition, inhibition of the process and activity of proteases and activators may occur. As a result, endothelial and epithelial cells rapidly undergo anoikis followed by inflammation and apoptosis when adhesion is disturbed [26,27]. The excessive growth of cancer cells is thus kept in check.
In addition, in silico molecular docking revealed the interaction between quercetin and eugenol with the active sites of VEGF, at positions Gln79, Pro49,    Lys48, Leu97, Glu38, and Asn75. VEGF is a very potent pro-angiogenesis factor in the growth process of cancer cells that modulate the proliferation and migration of cancer cells [28]. In lung cancer, VEGF is overexpressed and plays an essential role in cancer cells' growth and modulates other pro-angiogenic factors [12]. The binding between the active compounds in EEOS and the VEGF site through conventional hydrogen bonds is expected to inhibit the activity and stimulation. Our data are in line with the previous studies showing that the binding between VEGFB and VEGFR on the Pro143, Leu204, Phe172, Lys170, Pro173, Leu174, Lys171, Thr206, Glu208, Leu215, and Lys217 was able to inhibit interaction with the extracellular matrix. It is also known that silico molecular docking phytocompounds such as eriodyctiol, epicatechin, scutellarin, and ginkgolide A can bind to VEGF with tremendous energy as standard drugs for the 4T1 mammary carcinoma cell line [29,30]. We determined that the binding of eugenol and quercetin to VEGF resulted from the interaction of conventional hydrogen bonds and Pi-cation, Pi-sigma, and Pi-alkyl interactions, which significantly contributed to the stability of the bond structure [31][32][33][34]. Moreover, the binding interaction also supported the hydrogen's donor and acceptor abilities and thus may predict the antioxidant activity of quercetin and eugenol [16,35]. Furthermore, if EEOS was able to inhibit αvβ3, α5β1, and VEGF, would EEOS also be able to induce apoptotic activity? Based on our molecular docking, it was found that quercetin and eugenol were able to interact with the active sites of caspase-3 and caspase-9 proteins, Thr270, Arg241, Thr152, Gly153, Lys271, and Ile187 and Glu187, Glu259, and Leu240, respectively. The key to EEOS's pharmacological properties is its binding to the active site through conventional hydrogen bonds and Pi-cation, Pi-sigma, and Pi-alkyl interactions with amino acid residues in caspase-3 and caspase-9 proteins. Our in silico research found that quercetin and eugenol form stronger bonds in caspase-9 than caspase-3. Based on this binding, we postulated that EEOS could directly induce conformational changes in caspase-3 and caspase-9 to mature caspase-3 or cleaved caspase-3/caspase-9 to increase PARP's cleavage activity, followed by anoikis, which, in turn, initiates apoptosis. Our results were consistent with the previous studies using natural ingredients, namely, chalcone 9X, in silico, to induce conformational changes in caspase-8 and caspase-3 to initiate apoptosis [35].
In accordance with the results of the in silico molecular docking, we performed an in vitro analysis. The in vitro results were consistent with the in silico data showing that the main contents of EEOS, namely, flavonoids and eugenol, were able to inhibit cells' attachment to the extracellular matrix. EEOS may thus inhibit the adhesion of the NSCLC (A549) cells in the same way the conventional commercial drug cisplatin (used in this experiment) does. EEOS's mechanism of action may inhibit adhesion, invasion, and cell migration, thus triggering anoikis and apoptosis in the A549 cell line.

Conclusion
Our data revealed that EEOS could act as both an antiproliferative and apoptotic agent on NSCLC cells in in silico molecular docking and in vitro experiments. Nevertheless, further investigation of the mechanism, dosage, and other potential benefits of EEOS as a possible herbal medication to prevent NSCLC is needed.