Nicotiana glauca leaf extract exhibits synergistic antioxidant and anticancer activities revealed by phytochemical...

This study aimed to characterize the phytochemical composition and antioxidant, cytotoxic and molecular docking profile of Nicotiana glauca Graham (Solanaceae), a wild plant species naturalized along the Wadi El Natrun-North Coast Road, Egypt. N. glauca was selected from a preliminary screening of ten co-occurring wild plant species collected during the summer of 2022 on the basis of its highest extraction yield (2.20%), the richest qualitative phytochemical profile among the ten species, and its strong antioxidant activity. The aqueous ethanolic leaf extract of N. glauca and its sixteen column fractions were subsequently evaluated using four chemical antioxidant assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium permanganate (KMnO₄), and dichlorophenolindophenol (DCPIP). Comprehensive phytochemical characterization was performed using thin-layer chromatography (TLC), TLC-bioautography, Fourier-transform infrared spectroscopy (FTIR), mass spectrometry, ¹H and ¹³C nuclear magnetic resonance (NMR), and GC/MS/MS analyses. Total phenolic, flavonoid and alkaloid contents ranged from 0.3-8.4 g GAE/100 g DW, 0.1-2.14 g/100 g DW, and 0.31-7.01 g/100 g DW, respectively, across the extracts; a Pearson correlation analysis between these compound classes and antioxidant activity across the ten species did not reach statistical significance (p > 0.05), consistent with a ceiling effect at the tested concentration and supporting a combined rather than single-compound-class contribution to activity. GC/MS/MS analysis of fraction 11 revealed anabasine as the predominant alkaloid (39.26%), followed by nornicotine (36.50%). Two pure compounds were isolated and identified as N,N-methylenebis(14-docosene) (Compound A) and 14-methylhexadecanoic acid (Compound B). A synergistic interaction was observed between the crude extract and Compound A for both antioxidant (combination index, CI = 0.53) and anticancer activity (CI = 0.139-0.158), whereas an antagonistic effect was observed with Compound B (CI = 2.2-28.8). Both the crude extract and isolated compounds exhibited concentration-dependent cytotoxic activity against HeLa (IC₅₀ = 146.04 µg/mL) and HepG2 (IC₅₀ = 169.38 µg/mL) cancer cell lines. Treatment of HeLa cells with Compound A was associated with upregulation of the pro-apoptotic Bax gene, downregulation of the anti-apoptotic Bcl-2 gene, an increased Bax/Bcl-2 ratio, and elevated malondialdehyde (MDA) levels, findings consistent with, though not direct proof of, apoptosis induction and oxidative stress. Molecular docking analysis indicated that anabasine binds with favorable predicted affinity to the HPV16 E2 (PDB: 2Q79) and E6 (PDB: 7UAJ) oncoproteins; these computational results provide hypothesis-generating support for further exploration of anabasine against HPV-associated oncoproteins and require experimental validation. Overall, these findings characterize N. glauca as a promising wild source of antioxidant and cytotoxic bioactive metabolites and support further mechanistic and translational investigation.