Carly Eggers1, Masaya Fujitani2, Ryuji Kato3, Scott Smid4. 1. Discipline of Pharmacology, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, South Australia, Australia. 2. Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Japan. 3. Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Japan; Division of Micro-Nano Mechatronics, Institute of Nano-Life-Systems, Institute for Innovation for Future Society, Nagoya University, Japan. 4. Discipline of Pharmacology, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, South Australia, Australia. Electronic address: scott.smid@adelaide.edu.au.
Abstract
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 Aβ1-42 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 (Aβ1-42; 0-2 µM). Fluorescent staining was used to indicate effects of Aβ1-42 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 Aβ1-42 (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 Aβ1-42 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.
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 Aβ1-42 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 (Aβ1-42; 0-2 µM). Fluorescent staining was used to indicate effects of Aβ1-42 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 Aβ1-42 (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 Aβ1-42 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.
Authors: Janina K Bowen; Jacqueline M Chaparro; Alexander M McCorkle; Edward Palumbo; Jessica E Prenni Journal: Sci Rep Date: 2021-11-08 Impact factor: 4.379