Zafar, Mashal
(2024)
Evaluation of the anti-cancer potential of Nigella sativa and its bioactive constituents thymoquinone and alpha-hederin.
PhD thesis, University of Nottingham.
Abstract
The exploration of natural compounds with potential anti-cancer properties has gained significant attention in recent years. Nigella sativa (NS), commonly known as black seed, has emerged as a promising candidate due to its rich composition of bioactive constituents. Thymoquinone (TQ) and alpha-hederin (aH), the bioactive constituents of NS seeds, effectively inhibit tumour cell progression through diverse molecular mechanisms but their progression to clinical trials, like most plant derived therapeutic compounds, is confronted with two major challenges, their low bioavailability and toxicity at higher concentrations. The work herein began by investigating the anti-cancer activities of TQ and aH individually against cell lines of oesophageal, oral, breast and ovarian origin, specifically OE-19, H357, MCF-7, SK-OV-3 and MCF10A cells, each characterized by unique genetic and environmental factors. Both TQ and aH were shown to inhibit the proliferation, migration and cell viability in each cell line. To address the limitation of toxicity of TQ and aH, the effects of combination of both compounds at different percent concentrations were investigated in OE-19, H357, MCF-7 and SK-OV-3 cells where they demonstrated a high synergy in MCF-7 and SK-OV-3 cells, a moderate synergy in OE-19 cells and an antagonistic effect in H357 cells. Consequently, to address the second limitation regarding their low bioavailability a copolymer methyl ether-block-poly (lactide-co-glycolid-poly(ethylene glycol) (PLGA-PEG) nanoparticle (NP) vehicle for TQ, with further sodium dodecyl sulphate (SDS) modification (SDSNP0TQ) were developed (NP-TQ) which can improve the therapeutic index of a drug by readily penetrating biological membranes through their reduced particle size (79.30 ± 52 nm). Treatment of cells with NP-TQ and SDSNP-TQ significantly enhanced the antiproliferative activity of free TQ in each cell line by reducing the particle size, making TQ and aH more bioavailable to cancer cells.
The effects of thermal processing of NS seeds on their anti-cancer activity was next assessed as NS seeds are traditionally used as a spice for cooking where they are subjected to high temperatures before consumption. For this evaluation, a preliminary study was performed to identify ideal solvent to be used for the extraction of NS seeds. Neat and binary solvents, methanol and ethanol, were employed as solvents for the extraction of NS seed compounds and their antiproliferative effects were evaluated and compared using MTS assays. Methanolic extracts (Me-NS) showed significantly higher potency than ethanolic extracts (E-NS) of NS seeds due to which methanol was used as the extraction solvent in evaluating the effects of thermal processing of NS seeds at different temperatures. Methanolic extracts of seeds heated at different temperatures demonstrated increased antiproliferative activity when heated at 50 °C and 75 °C, moderate antiproliferative activity when heated at 100 °C, mild antiproliferative activity when heated at 150 °C and a low antiproliferative activity when seeds were heated at 200 °C prior to extraction, providing salient insights into the importance of the relationship between the potency of NS seed extracts and thermal processing of the seeds. To identify the metabolic changes associated with the thermal processing of NS seed extracts, extracts heated at each time point and control (with no thermal processing) were modified for untargeted LC-MS metabolic analysis.
Finally, an untargeted LC-MS method was used to identify the intracellular pathways that could suggest the full pharmacological mechanism of action of TQ and aH related to cell death in OE-19, H357, MCF-7 and SK-OV-3 cells. Due to the national lockdown associated with the Covid-19 pandemic, periodic breakdown of Q-Exactive LC-MS machine and software, and delays in delivery of experimental reagents, the complete metabolomic analysis of all variables of NS extracts (Me-NS, E-NS and NS extracts of thermally processed seeds) was not completed. Multivariate analysis of different signals detected from the intracellular samples of OE-19, H357, MCF-7 and SK-OV-3 cells treated with TQ and aH demonstrated a clear difference between each tested group and the comparative OPLS-DA score plots of cell extracts following treatment with TQ, aH or appropriate media alone showed a clear separation between each treated and untreated group. R2 and Q2 values ≥ 0.5 demonstrated an acceptable OPLS-DA model that is worth future studying to identify key metabolites responsible for the separation in the OPLS-DA models and reveal several affected pathways modulated by TQ and aH for their anti-cancer activity using pathway analysis tools.
In conclusion, this thesis provides evidence that the limitations of NS seed constituents can be overcome by combination therapies and using PLGA-PEG nanocarriers to minimize toxicity and enhance their activity and can be regarded as propitious evidence for the progression of NP-TQ for in vivo studies followed by clinical trials. Furthermore, the biological activity of NS seed extracts in relation to thermal processing provides important insight that would affect the everyday personal use of NS seeds. SK-OV-3 cells were concluded to be the most sensitive to the effects of NS seed extracts and their individual constituents and H357 cells were the least sensitive cells.
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