Novel cannabis flavonoid, cannflavin A displays both a hormetic and neuroprotective profile against amyloid β-mediated neurotoxicity in PC12 cells: Comparison with geranylated flavonoids, mimulone and diplacone

Background: including inhibition of amyloid beta (A beta) fibrillisation and neurotoxicity of relevance to Alzheimer's disease. Cannabis contains a unique subset of prenylated flavonoids, the cannflavins. While selected conventional flavonoids have demonstrated anti-amyloid and neuroprotective potential, any neuroprotective bioactivity of prenylated flavonoids has not been determined. We evaluated the in vitro neuroprotective and anti-aggregative properties of the novel geranylated cannabis-derived flavonoid, cannflavin A against A beta 1-42 and compared it to two similarly geranylated flavonoids, mimulone and diplacone, to compare the bioactive properties of these unique flavonoids more broadly. Methods: Neuronal viability were assessed in PC12 cells biochemically using the MTT assay in the presence of each flavonoid (1-200 mu M) for 48 h. Sub-toxic threshold test concentrations of each flavonoid were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-bhp) or amyloid beta (A beta 1-42; 0-2 mu M). Fluorescent staining was used to indicate effects of A beta 1-42 on PC12 cellular morphology, while direct effects of each flavonoid on A beta fibril formation and aggregation were assessed using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualise fibril and aggregate morphology. Results: Cannflavin A demonstrated intrinsic hormetic effects on cell viability, increasing viability by 40% from 1 to 10 mu M but displaying neurotoxicity at higher (> 10-100 mu M) concentrations. Neither mimulone nor diplacone exhibited such a biphasic effect, instead showing only concentration-dependent neurotoxicity, with diplacone the more potent (from > 1 mu M). However at the lower concentrations (< 10 mu M), cannflavin A increased cell viability by up to 40%, while 10 mu M cannflavin A inhibited the neurotoxicity elicited by A beta 1-42 (0-2 mu M), reducing A beta aggregate adherence to PC-12 cells and associated neurite loss. The neuroprotective effects of cannflavin A were associated with a direct inhibition of A beta 1-42 fibril and aggregate density, evidenced by attenuated ThT fluorescence kinetics and microscopic evidence of both altered and diminished density of A beta aggregate and fibril morphology via electron microscopy. Conclusions: These findings highlight a concentration-dependent hormetic and neuroprotective role of cannflavin A against A beta-mediated neurotoxicity, associated with an inhibition of A beta fibrillisation. The efficacy of the cannabis flavone may itself direct further lead development targeting neurodegeneration in Alzheimer's disease. However, the geranylated flavonoids generally displayed a comparatively potent neurotoxicity not observed with many conventional flavonoids in vitro.