Chemical composition, in vitro and in silico evaluation of essential oil from Eucalyptus tereticornis leaves for lung cancer

Abstract Chemical composition of the essential oil (EO) of Eucalyptus tereticornis leaves was studied by gas chromatography–mass spectrometry. Forty-five constituents were identified in the oil hydrodistilled from the sample collected from Ghudda Village, Bathinda (Pb), India of which eucalyptol (34.39%) and ledol (9.92%) were the major constituents. In vitro antioxidant and anticancer potential of EO was analysed by DPPH 2,2-diphenylpicrylhydrazyl (DPPH) and MTT assay. The percentage free radical scavenging activity was found to be 63.77%. The antiproliferative activity was analysed using MTT assay in adenocarcinomic human alveolar basal epithelial A549 cancer cell line and showed IC50 value of 47.14 µg/ml. In silico study of EO, constituents were performed using Maestro 12.9 against EGFR (PDB ID-2RGP). Five constituents from EO showed high dockscore as compared to standard Mobicertinib which indicated the effectiveness of oil constituents against lung cancer.


Introduction
Malignant tumours are major public health concern among which the most prevalent is lung cancer (Rybarczyk-Kasiuchnicz et al. 2021;Wen et al. 2021). Lung cancer has the greatest mortality rate, making it the main cause of death. According to GLOBOCAN 2020, lung cancer was the second most commonly diagnosed carcinoma and was responsible for 2.21 million cases worldwide. It remained the leading cause of cancer death (18%) with an anticipated 1.8 million from lung cancer (Sung et al. 2021). Approximately 98% of lung cancers are carcinomas or tumors derived from transformed cells of epithelial lineage. Recently, nearly four dozen different histopathological variants of lung carcinoma have been recognized. Lung cancer are generally divided in to two categories: small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). About 85% lung cancers are NSCLC (Wen et al. 2021;Zamay et al. 2017). The epidermal growth factor receptor (EGFR) was recently shown to play a pivotal role in tumorigenesis which is expresses in more than 60% of NSCLCs (da Cunha Santos et al. 2011). The EGFR inhibitors are considered an emerging class of targeted therapeutic agents for the treatment of NSCLCs. Although most EGFR-mutant NSCLCs initially respond to EGFR inhibitor, such as gefitinib (Iressa) and erlotinib (Tarceva), the vast majority of these tumors ultimately become resistant to drug treatment (Zarogoulidis et al. 2015;Bencardino et al. 2007).
Various natural products derived phytoconstituents are being tried for anti-cancer potential due to their unique structural, chemical, and biological variety have traditionally played an important role in cancer treatment with minimal side effects (Banerjee et al. 2015;Newman and Cragg 2020;Atanasov et al. 2021;Porras et al. 2021). Eucalyptus is the largest aromatic genus and belongs to the Myrtaceae family having 900 species and subspecies in the genus (Barbosa et al. 2016). Eucalyptus tereticornis is a tree that grows up to 50 m tall. This species is one of the main sources of EO's used for medicinal purposes. In previous studies, the EO of Eucalyptus was shown to exert various pharmacological activities, including CNS stimulant, analgesic, antiviral, antibacterial, wound healing, pain management, in respiratory disorders and anticancer (Kiran et al. 2020;Chandorkar et al. 2021). In present study we evaluated the chemical composition, In vitro antioxidant and antiproliferative activities followed by in silico estimation of their affinity towards EGFR (da Cunha Santos et al. 2011).

In vitro studies
In vitro antioxidant activity of eucalyptus oil was evaluated by DPPH radical scavenging assay. EO has shown 63.77% radical scavenging activity with respect to standard ascorbic acid. EO was also evaluated for antiproliferative activity using MTT assay in adenocarcinomic human alveolar basal epithelial A549 cancer cell line and showed potent activity with IC 50 value of 47.14 mg/ml at 24 h treatment (Figure 1). High content of oxygenated mono-and sesquiterpenes may be responsible for the activity of essential oil. Some of the recent studies has also reported the antioxidant and cytotoxic activity of the Eucalyptus spp. The cytotoxicity has been reported on Breast cancer cell lines (MCF-7 cell) line which indicate the effectiveness of oil as anticacer (Singh et al. 2009;Kiran et al. 2020).

In silico study of essential oil constituents
Phytoconstituents of essential oil identified via. GC-MS were also evaluated for their anti-lung cancer activity through in silico approach. It was reported that endothelial growth factor receptor (EGFR) is overexpressed in lung cancer cell lines. Molecular docking studies was performed to understand the binding intereaction of the constituents with EGFR (PDB ID 2RGP) and compared with standard Mobocertinib. The docking process was validated by calculating root mean square deviation (RMSD) which was found to be 0.649 Å ( Figure S3). It was observed that many constituents have dock score better than that of standard drug which implies that they have good binding affinity towards EGFR (Table S2) with 2-Methylbutanoic anhydride has the highest dock score (À6.13 kcal/mol) while Mobocertinib has dock score of À3.55 kcal/mol. Moreover, other physicochemical parameters such donorHB, accptHB, dipole and volume were also within the required range. Also, constituents were binding in the same cavity as suggested by amino acid residues interaction. Standard drug and 2-Methylbutanoic anhydride were interacting with Met793 through hydrogen bonding, while both shows van de waal interactions with Thr790, Gln791, and Thr854 ( Figure  S4). All other hit molecules trans-Linalool oxide, 1(2H)-Naphthalenone, octahydro-7, 1,3,3-Trimethyl-2-vinyl-1-cyclohexene, Carvacrol) showed a similar binding pattern as that of standard within the binding site pocket.
The stability of protein ligand complex was also evaluated though their binding free energy calculations through MM-GBSA. The binding free energies of top five hit molecules and standard drug mobocertinib are shown in Table S3. Based on the given data, it is feasible to conclude that MMGBSA dG Bind vdW terms are the primary energy aspects that improve binding, as well as docking posture optimization. Moreover, rescoring stronger complexes arise when free binding energy is negative. As a result, ligands with low free binding energy have greater affinity for target receptor than ligands with high free binding energy.
Pharmacokinetic properties play an important role in drug discovery process. In present study various pharmacokinetic parameters for the top five hit molecules were also predicted using QikProp module of the Schrodinger suite (Table S4). It was observed that the top components of EO were found to exhibit 100% human oral absorption while two compounds 2-Methylbutanoic anhydride and 1,3,3-trimethyl-2vinyl-1-cyclohexene were showing 94.352% and 94.604% human oral absorption respectively. The partition coefficient octanol/water values for the top hit compounds were predicted to be within the range which indicates that these ligands possessed balanced lipophilicity and hydrophobicity. Likewise other parameters such as QPPCaco, QPlogKhsa, QPlogBB and QPlogKp were all within the required range for all the compounds.

Experimental
Please refer supplementary information file for the procedures of methods and assays used in present study.

Conclusion
Essential oils offer a wide range of therapeutic potential, including anticancer activity, and have been used for centuries to cure a variety of human ailments and diseases. In present study, chemical composition of EO extracted through hydrodistillation was determined using GC-MS. Forty five constituents were identified with eucalyptol having highest percentage of 34.39%. EO was further evaluated in vitro for its antioxidant and antiproliferative activity and found to have 63.77% radical scavenging activity along with IC50 value of 47.14 mg/ml against adenocarcinomic human alveolar basal epithelial A549 cancer cell line. Chemical constituents of the eucalyptus oil were also analysed by in silico study for their affinity towards EGFR along with a standard drug (Mobocertinib). Among the various constituents 2-Methylbutanoic anhydride was observed to have best dock score of À6.13 kcal/mol. ADME profile of the constituents of EO was also analysed and found to be with in the range which indicated the effectiveness of oil constituents against lung cancer.