Exploring the Bioactivity and Therapeutic Potential of Structurally Diverse Phytochemicals in Neurodegenerative and Gastrointestinal Disease

Abstract

Cannabis sativa is the source of over 540 natural products, including phytocannabinoids, terpenoids and flavonoids. These natural products have been ascribed protective properites in various contexts, including neurodegenerative and gastrointestinal diseases. However, the pharmacological diversity of Cannabis is yet to be truly explored, offering exciting potential in the treatment of such diseases. Alzheimer’s Disease (AD) is the most common neurodegenerative disease, characterised by the progressive loss of cholinergic neuronal structure and synaptic function. The pathological hallmark of AD is the aggregation of amyloid β (Aβ) and its associated neurotoxicity, which result in this neruonal loss. Inflammation and oxidative stress are crucial factors not only in the progression of AD but also in inflammatory bowel disease (IBD) and gastrointestinal mucositis (GIM), resulting in reduced epithelial barrier function. Therefore, a drug which reducess the cytotoxicity of Aβ, maintains barrier function and possesses anti-inflammatory and antioxidant properties may offer wide-ranging benefits in the amelioration of AD, IBD and GIM. Treatment of PC12 cells with Aβ results in significant loss of cell viability in the MTT assay, while also inducing key changes in neuronal cell morphology as determined by fluorescence microscopy. We identified novel bioactivity for the phytocannabinoids cannabigerol (CBG), cannabinol (CBN) and cannabichromene (CBC), which effectively prevented this loss of viability in a manner independent of either antioxidant capacity or direct interactions with Aβ, while also maintaining healthy cell morphology in the presence of Aβ. Δ9-tetrahydrocannabinol (Δ9-THC) significantly inhibited Aβ neurotoxicity, while cannabidiol (CBD) proved an effective antioxidant with no significant effect on Aβ-evoked neurotoxicity; cannabidivarin (CBDV) reduced neuritic projections in PC12 cells without affecting cell viability. To assess the neuroprotective capacities of whole botannical cannabis extracts, in comaprison with pure phytocannabinoids, five proprietary chemovars were selected. The Δ9-THC-dominant chemovars BC-101, BC-201 and BC-401 prevented Aβ-induced neurotoxicity in NSC-34 cells without inhibiting tert-butyl hydroperxoide (tbhp)-induced lip peroxidation; no significant protection was observed following treatment with the CBD-dominant chemovars BC-301 or BC-501; heating of each extract reduced their neuroprotective capacity. The final two studies investigated phytocannabinoids and flavonoids as inhibitors of cytokine and 7-ethyl-10-hydroxycamptothecin (SN-38)-evoked decreases in epithelial barrier function in Caco-2 cells. We identified novel roles for CBD, CBG and CBDV as inhibitors of SN-38- evoked increases in permeability, with each phytocannabinoid screened, including CBC, inhibiting cytokine-evoked increases in permeability; no antioxidant capacity was observed in the DCFDA assay. A novel role was identified for the flavonoid 2-D08 as a dual inhibitor of cytokine and SN-38-induced permeability increases, with novel protection also demonstrated for the flavonoids myricetin and transilitin against SN-38; each flavonoid significantly inhibited reactive oxygen species generation in the DCFDA assay. In conclusion, this thesis demonstrates novel protective roles for phytocannabinoids as inhibitors of Aβ-evoked neurotoxicity and changes in neuronal morphology, as well as novel roles for select phytocannabinoids and flavonoids as inhibitors of cytokine and SN-38-evoked reductions in epithelial barrier function. These findings highlight the wide-ranging potential of natural products as protective agents in the context of AD, IBD and GIM, highlighting novel lead candidates for drug development targeting these conditions.