Characterisation of fucoidan extracts from seaweed varieties : implications for treating inflammatory bowel diseases
Creators: name: Ahmad, Tauseef
Publisher: name: University of Tasmania
Year: 2024

description: Inflammatory bowel diseases (IBD), such as Crohn's disease and Ulcerative Colitis, are long-term, uncontrolled chronic inflammatory conditions of the gastrointestinal tract commonly present in adolescence and early adulthood. The trajectory of IBD typically includes periods of remission, exacerbations, and relapses, although they are not the only possible outcomes. The gastrointestinal tract becomes red, bloated, painful, and dysfunctional and is associated with various complications, including diarrhoea, bleeding, extreme exhaustion, weight loss, anaemia, and bowel obstruction. The IBD aetiology is multifactorial, involving a complex interaction among genetic susceptibility, environmental triggers, and immunological and infectious (bacterial) factors and deciphering the interactions among these factors to get relevant data remains a formidable challenge. The condition can become severe enough to necessitate hospitalization, and many individuals with IBD require surgery. <br>Despite the availability of pharmacotherapies such as corticosteroids, immunosuppressants, and aminosalicylic acid medications, inflammatory bowel disease (IBD) continues to exhibit high long-term therapy failure rates, often with adverse effects. The escalating prevalence of IBD poses substantial financial burdens on healthcare systems in developed and developing countries. Identifying novel therapeutic targets and consistently refining current approaches to optimise treatment response rates, safety profiles, and drug tolerance is critical. <br>The novel drug discovery initiative seeks bioactive compounds within medicinal plants targeting diverse pharmacological objectives, notably addressing IBD. Among emerging therapeutic agents, Fucoidans stand out for their prospective efficacy in treating IBD and related inflammatory conditions. Fucoidans, fucose-rich sulfated polysaccharides, are found within cell walls of multiple brown seaweed species, such as <i>Undaria pinnatifida</i> (wakame), <i>Fucus vesiculosus</i> (bladderwrack), <i>Cladosiphon sp</i>. (mozuku), and <i>Laminaria japonica</i> (kombu).<br>Fucoidans encompass a spectrum of pharmacological attributes, spanning anti-inflammatory, antitumor, antioxidant, anticoagulant, immunomodulatory, and antiviral activities. Notably, their anti-inflammatory effects encompass complement inhibition, selectin inhibition, and enzyme suppression. Moreover, fucoidans orchestrate immune cell modulation, affecting macrophages, natural killer cells, dendritic cells, and lymphocytes while downregulating key pro-inflammatory cytokines like TNF-α, IL-1β, and IL-6. However, the therapeutic potency of fucoidans is contingent upon factors such as seaweed species, geographic origin, molecular structure, weight, and extraction protocols. Based on these reasons, my study aim was twofold: Firstly, to comprehensively investigate and characterise the relative effect of a wide range of fucoidan extracts, including higher and lower molecular weights, as well as crude and depyrogenated extracts with a primary focus on assessing their anti-inflammatory and immunomodulatory properties using cell models. Secondly, to investigate the therapeutic potential of fucoidan extracts (UPF, FVF, MPF and DP-MPF) in a mouse model of colitis to identify the most potent fucoidan extracts exhibiting significant anti-inflammatory and antioxidant properties in an animal model of colitis. <br>Specifically, in study 1 (Thesis chapter 2), various extracts of fucoidan obtained from different seaweed sources: <i>Undaria pinnatifida</i> (UPF), <i>Fucus vesiculosus</i> (FVF), <i>Macrocystis pyrifera</i> (MPF), <i>Ascophyllum nodosum</i> (ANF), and <i>Laminaria japonica</i> (LJF) were tested and investigated for immunomodulatory properties including inhibition and modulation of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in human peripheral blood mononuclear cells (PBMCs) and human monocytic cell line (THP-1) in lipopolysaccharide (LPS) induced conditions. LPS is a gram-negative bacteria cell wall component often used to trigger systemic inflammation. My data indicate that none of the fucoidan extracts demonstrated any signs of cytotoxicity in human THP-1 cells after incubation of 48 h using an MTT (3- [4,5-dimethylthiazol-2-yl]-2,5, -diphenyltetrazolium bromide) cell viability assay. Additionally, in LPS-induced human THP-1 and PBMC cells, all fucoidan extracts significantly downregulated pro-inflammatory cytokines TNF-α and IL-1β levels in a dose-dependent fashion. Mainly, <i>Macrocystis pyrifera</i>-derived DP-MPF was a highly effective inhibitor at lower concentrations. The insights gained from examining various fucoidan extracts offer valuable understanding regarding potential strategies to enhance their efficacy against inflammation-related pathology and provide a solid platform for the subsequent <i>in-vivo</i> experiments in the project. <br>Drawing upon the results from the <i>in-vitro</i> studies, in study 2 (Thesis chapter 3), I investigated <i>Undaria pinnatifida</i> (UPF) and F<i>ucus vesiculosus</i> (FVF) crude extracts immunomodulatory potential in a dextran sulfate sodium (DSS) colitis mouse model. DSS was administered in drinking water to induce symptoms of acute colitis in male C57BL/6 mice, including significant epithelium disruption and a substantial inflammatory reaction in the colon. DSS-treated mice exhibit acute colitis symptoms such as rectal bleeding, body weight loss, and diarrhoea. Oral treatment with UPF and FVF extracts at 1mg/day exhibited significant therapeutic effects in alleviating colitis symptoms. UPF treatment resulted in reduced diarrhoea (p&lt; 0.0175), decreased faecal blood loss (p&lt; 0.0229), preserved body weight (p&lt; 0.0004), decreased colon weight (p&lt; 0.0191), reduced spleen weight (p&lt; 0.0146), and maintained colon length (p&lt; 0.0094). Similarly, FVF treatment demonstrated remarkable outcomes, showing significant reductions in diarrhoea (p&lt; 0.0010), faecal blood loss (p&lt; 0.0001), retention of body weight (p&lt; 0.0001), colon weight (p&lt; 0.0020), spleen weight (p&lt; 0.0004), and colon length preservation (p&lt; 0.0001). These findings underscore the potential of UPF and FVF extracts as effective interventions for mitigating the symptoms associated with colitis. Furthermore, the histological evaluation revealed that UPF and FVF effectively reduced colitis-associated pathological parameters, including the prevention of crypt architecture loss and goblet cell reduction, as well as a decrease in immune cell infiltration and oedema (p&lt; 0.0032 and p&lt; 0.0003 respectively). Additionally, the macroscopic changes induced by UPF and FVF correlated significantly with substantially reduced production of at least 18 pro-inflammatory cytokines in colon tissue. My study results demonstrated that UPF and FVF provide significant protection against acute colitis by significantly reducing clinical symptoms of colitis, retaining the macroscopic architecture of the colon induced by colitis and reducing the production of pro-inflammatory cytokines in the colon, including TNF-α, IL-1β, and IL-6, IL?10, IFN-γ, MIP-1α, MIP-1β, G-CSF and GM-CSF. <br>Similarly, in Study 3 (Thesis Chapter 4), I examined the crude and low molecular weight depyrogenated (DP-MPF) extracts of <i>Macrocystis pyrifera</i> (MPF) to assess their cytotoxicity, anti-inflammatory potential, and efficacy in DSS-induced colitis mouse model. Hence, both extracts were initially assessed using an <i>in-vitro</i> model involving LPS-induced RAW 264.7 macrophage cell lines to gauge their anti-inflammatory effects by inhibiting major pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). In the study's second phase, both extracts were tested in the DSS-induced colitis mouse model for their effectiveness and protective effects in colitis. Fucoidan extracts from MPF and DP-MPF displayed no cytotoxic effects in LPS-induced mouse RAW 264.7 macrophage cell lines. Additionally, we demonstrated that MPF and DP-MPF exert anti-inflammatory properties by downregulating major pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in the macrophages. <br>Furthermore, MPF and DP-MPF demonstrated that these extracts are highly potent in the acute colitis model. Both extracts have reduced the clinical symptoms of colitis by reducing the body weight loss (p&lt; 0.0002, p&lt; 0.0001), reducing the blood stool score (p&lt; 0.0013, p&lt; 0.0001) and retaining the stool consistency (p&lt; 0.0023, p&lt; 0.0001) respectively. Additionally, MPF and DP-MPF treatment significantly protected the changes to the spleen weight (p&lt; 0.0185, p&lt; 0.0003), colon weight (p&lt; 0.0076, p&lt; 0.0001) and colon lengths (p&lt; 0.0195, p&lt; 0.0001) respectively. The histopathological evaluation demonstrated significantly lower pathological scores in MPF and DP-MPF treated groups (p&lt; 0.0063, p&lt; 0.0001). Most importantly, in this study, in line with the macroscopic findings, fucoidan extracts showed significant inhibition of at least 18 pro-inflammatory cytokines reported to be at higher levels in colitis conditions. This study established the safety profile of <i>Macrocystis pyrifera</i> extracts (MPF, DP-MPF) for the first time. Both extracts protect against acute colitis symptoms, including body weight loss, diarrhoea, and rectal bleeding. Protect the structural integrity and architecture of the colon and inhibit colonic inflammation by reducing the pro-inflammatory cytokine production, including TNF-α, IL-1β, and IL-6, IL-10, IFN-γ, MIP-1α, MIP-1β, G-CSF and GM-CSF. In addition, both MPF and DP-MPF significantly reduced the colonic levels of myeloperoxidase (MPO), nitric oxide (NO), and malondialdehyde (MDA) a. They increased the levels of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) in DSS-induced colitis mouse model.<br>In summary, these findings strongly support fucoidan's potential as a ...