Phytochemical and Antioxidant analysis
Extraction of Balanites aegyptiaca Linn. Delile is done by four different organic solvents i.e., heptane, hydro-alcohol, acetone, and ethyl acetate. The qualitative analysis of the extract of all solventsis shown in Table 1. Saponins show antioxidant, antimicrobial, and anti-inflammatory properties [19].
Flavonoids exhibit antioxidant and anti-inflammatory propertytannin exhibits antimicrobial and free radical scavenging activity [20, 21]. Steroids show anti-inflammatory properties [22].It was already documented that the major classes of antimicrobial compounds from plants have tannins, saponins, flavonoids, and glycosides [23].
The repeated purification of ethyl acetate fraction over the silica-gel column led to the isolation of Balanitin 6, a steroidal saponin. The structure of Balanitin 6 (Fig. 3) was confirmed by NMR spectroscopy. The obtained NMR spectra were compared with the reported NMR and both were in complete agreement thus, the isolated compound was confirmed as Balanitin 6 [24]. The NMR spectra of Balanitin 6 are provided in the Supplementary material (Fig. S1-S4).
Cell viability
The cytotoxicity of plant extract in MCF-7, MDA-MB 231, FaDu, and PBMCs cells was evaluated using an MTT assay and significant (***p≤0.001, **p≤0.01 and *p≤0.05) dose-dependent decrease in cell viability was observed in breast cancer cell lines (MCF-7, MDA-MB 231), head and neck cancer (FaDu) lines at 24 and 48h whereas, plant extract has minimal effect on PBMCs (Fig.4). This anti-cancer activity of plant extract might be due to the presence of flavonoids, and phenolic compounds observed in the preliminary test (Table 2). Further NMR study of Balanites aegyptiaca plant with fresh and dry fruit in our previous study reported the presence of compounds like oleic acid, linoleic acid, and linolenic acid [25], which has been reported to have an anti-tumorigenic effect [26, 27].
Plant extract treatment Decreases cell Migration
To study the effect of plant extract on cell migratory ability using a scratch assay and the results demonstrated that the mean scratch distance was found to be significantly larger (Fig.5) with the increasing dose of plant extract (Fig. 2) quantified by image J this may be due to the presence of phytochemicals present in the plant extract that inhibit the migratory ability of breast cancer cells. A similar result was observed by Hsu et al.,2015 where the breast cancer cells’ wound area was significantly larger than to control with the increasing dose of betulin [14].
Molecular Docking
The Balanitin-6 showed important interactions with the protein viz. H-bond and π-alkyl. The binding energy, ligand efficiency, and interactions of Balanitin-6 and Gefitinib (standard) with EGFR (PDBid. 6DUK) are presented in Table 2. The Balanitin-6 showed good binding energy and ligand binding efficiency which was -10.438 kcal/mol and -0.168 kcal/mol, respectively and its binding energy was comparable to Gefitinib. Balanitin-6 showed H-bond interaction with Lys745, Asp837, and Asp855. The 2D and 3D interaction diagrams of these two compounds have been represented in Fig.6.