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: Flavonoids form a diverse class of naturally occurring Polyphenols ascribed various biological activities, including inhibition of amyloid β (Aβ) 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 Aβ_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 µM) 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 β (Aβ_1-42; 0-2 µM). Fluorescent staining was used to indicate effects of Aβ_1-42 on PC12 cellular morphology, while direct effects of each flavonoid on Aβ 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 µM but displaying neurotoxicity at higher (>10-100 µM) concentrations. Neither mimulone nor diplacone exhibited such a biphasic effect, instead showing only concentration-dependent neurotoxicity, with diplacone the more potent (from >1 µM). However at the lower concentrations (<10 µM), cannflavin A increased cell viability by up to 40%, while 10 µM cannflavin A inhibited the neurotoxicity elicited by Aβ_1-42 (0-2 µM), reducing Aβ aggregate adherence to PC-12 cells and associated neurite loss. The neuroprotective effects of cannflavin A were associated with a direct inhibition of Aβ_1-42 fibril and aggregate density, evidenced by attenuated ThT fluorescence kinetics and microscopic evidence of both altered and diminished density of Aβ aggregate and fibril morphology via electron microscopy.

Conclusions: These findings highlight a concentration-dependent hormetic and neuroprotective role of cannflavin A against Aβ-mediated neurotoxicity, associated with an inhibition of Aβ 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.

Amyloid β; Cannflavin A; Diplacone; Hormesis; Mimulone; Neuroprotection.