Chemical Profiling and Investigating the Anti-Inflammatory Properties of Polyphenol Enriched Sugarcane Extracts

Sugarcane (Saccharum officinarum), a globally significant crop primarily cultivated for sugar and ethanol production, generates large volumes of polyphenol-rich by-products. These by-products hold promising bioactive potential, particularly due to their polyphenol content, which exhibits antioxidant and immunomodulatory properties. This thesis presents a comprehensive investigation into the chemical composition and anti-inflammatory effects of Polyphenol-Rich Sugarcane Extracts (PRSEs), through a multidisciplinary approach integrating extraction optimisation, in vitro immunological assays, and mass spectrometry-based chemical profiling techniques. The study commenced with the optimisation of extraction methods to enhance the recovery of total polyphenols and flavonoids from sugarcane-derived materials. A comparative assessment of liquid-liquid extraction (LLE) solvents and post-extraction strategies identified ethyl acetate, coupled with acidification and solid-phase extraction (SPE), as the most effective approach to yield PRSEs with high antioxidant capacity, as demonstrated by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Cytotoxicity analyses using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), Trypan Blue, and CellTox™ assays across myeloid-derived cell lines (THP-1, U937, and dHL-60) confirmed that the PRSEs were well tolerated at varying doses, though high concentrations interfered with tetrazolium-based assays, necessitating cross-validation with orthogonal methods. Subsequent immunological evaluations revealed that PRSEs exhibit both antioxidant and immunomodulatory activities under different stimulation conditions. In baseline and LPS-stimulated settings, the extracts reduced intracellular reactive oxygen species (ROS) and altered cytokine profiles. Specifically, Polyphenol Rich Sugarcane Extracts (PRSEs) significantly suppressed Interleukin (IL)-6 secretion in post-Toll-Like Receptor (TLR)4 stimulated THP-1 cells, while exhibiting dose- and context-dependent effects on TNF (Tumour Necrosis Factor)-α. Pre-treatment with extracts prior to inflammatory stimulation demonstrated contrasting responses, indicating complex temporal dynamics in polyphenol-cell interactions. Furthermore, gene expression analysis using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) showed minimal changes in cytokine transcription, suggesting that PRSEs exert their effects predominantly at the post-transcriptional or secretory level. To establish the chemical basis of these biological effects, high-resolution mass spectrometry was employed to characterise and quantify polyphenols present in PRSEs. An in-house polyphenol standard library was created, encompassing a broad panel of flavonoids and phenolic acids. Using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS), key polyphenols were identified, including swertisin, tricin, chlorogenic acid, syringic acid, and trans-p-coumaric acid. Method validation confirmed the mass accuracy and precision of the LC-QTOF-MS approach. Targeted quantification using tandem mass spectrometry (LC-MS/MS) was then developed and validated for trans-p-coumaric acid, achieving accurate and precise quantification across both Polynol (PN) and Polygain (PG) extract variants. Overall, this research provides novel insights into the dual chemical and biological profiles of sugarcane polyphenol extracts. The findings demonstrate that PRSEs, derived from sugarcane waste streams, are not only chemically rich in bioactive flavonoids and phenolic acids but also capable of modulating inflammation and oxidative stress responses in immune cells. This work contributes to the growing body of evidence supporting the therapeutic and nutraceutical applications of agricultural by-products, offering potential pathways for sustainable bioproduct development and functional food design. Future directions include in vivo validation, exploration of bioavailability, and formulation into dietary supplements or clinical interventions for chronic inflammatory conditions.<p></p>