Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf

Abstract

Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.

Potent antioxidant and anticancer activities of the methanolic extract of Calligonum comosum (L’Her) fruit hairs against human hepatocarcinoma cells

“…For the cytotoxicity assessment, cells were trypsinized (Trypsin 0.05%/0.53 mM EDTA). Then, cells were seeded (2 × 10 3 cell/well in 100 μl of medium) into 96-well plates (Gibco, USA) for 24 h before treated with different concentrations of MeCc (2.5, 5, 7.5, 10, 15, 20 and μg/ml) for another 24 h ( Aboul-Soud et al, 2020 , Abo-Salem et al, 2020 ). To analyze the gene expression level and western blot of pro- and anti-apoptotic markers (p53, Bax, Caspase 3 and Bcl-2) , HepG-2 and BJ-1 cells were seeded (6 × 10 6 cell/well in 3 ml of medium) into in a 6-well plate.…”

“…After 24 h of incubation with different MeCc concentrations, the media were discarded, and adherent cells were incubated with 100 µl/well MTT at a concentration of 0.5 mg/ml prepared in PBS and subsequently incubated at 37 °C for additional 3 h at 37 °C under dark condition ( Aboul-Soud et al, 2020 , Mosmann, 1983 ). Then, 100 μl isopropyl alcohol was added per well in order to dissolve the purple formazan crystals by the help of shaking for another 2 h at room temperature.…”

“…The results were analyzed in triplicates and the viability percentage were calculated. The MeCc concentrations resulting in 50% growth inhibition of HepG2 cells (IC 50 ) were calculated by use of Microsoft Excel trendline equation ( Aboul-Soud et al, 2020 ).…”

“…The RT-qPCR reactions were performed in a thermal cycler 7500 Real-Time PCR system (Applied Biosystems) and the amplification data was analyzed by the 7500-software version 2.0. PCR conditions were: 2 min at 50° C, 10 min at 95° C, and 40 cycles of 15 s at 95° C and 30 s at 60° C. The 2 −ΔΔCt method was used to determine the relative mRNA expression level for a specific gene and were represented as fold change compared to β-actin, as a house-keeping gene ( Aboul-Soud et al, 2020 ).…”

Lycopene nanoparticles ameliorate the antioxidant, antimicrobial and anticancer potencies of tomato pomace.

“…Cell lines were cultured in DMEM/high glucose media supplemented with 2 mM L-glutamine, 10% FBS and 1% penicillin/streptomycin kept in Corning ® 75cm² U-Shaped canted neck cell culture flask with vent cap (Corning, New York, USA). Then, sub-confluent cultures (70-80%) were trypsinized (Trypsin 0.05%/0.53 mM EDTA) and spilt depending on the seeding ratio [26,27].…”