Characterizing cannabis-prevalent terpenes for neuroprotection reveal a role for α and β-pinenes in mitigating amyloid β-evoked neurotoxicity and aggregation in vitro.

BACKGROUND: Cannabis sativa L. (C. sativa) can efficiently synthesize of over 200 terpenes, including monoterpenes, sesquiterpenes and triterpenes that may contribute to the known biological activities of phytocannabinoids of relevance for the burgeoning access to medicinal cannabis formulations globally; however, to date have been uncharacterized. We assessed twelve predominant terpenes in C. sativa for neuroprotective and anti-aggregative properties in semi-differentiated PC12 neuronal cell line that is robust and validated as a cell model responsive to amyloid β (Aβ1-42 ) protein exposure and oxidative stress.

METHODS: Cell viability was assessed biochemically using the MTT assay in the presence of myrcene, β-caryophyllene, terpinolene, limonene, linalool, humulene, α-pinene, nerolidol, β-pinene, terpineol, citronellol and friedelin (1-200 μM) for 24 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP; 0-250 μM) or amyloid β (Aβ1-42 ; 0-1 μM) to assess neuroprotective effects. Direct effects of each terpene on Aβ fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualize fibril and aggregate morphology.

RESULTS: Terpenes were intrinsically benign to PC12 cells up to 50 μM, with higher concentrations of β-caryophyllene, humulene and nerolidol inducing some loss of PC12 cell viability. No significant protective effects of terpenes were observed following t-BHP (0-200 µM) administration, with some enhanced toxicity instead demonstrated from both β-caryophyllene and humulene treatment (each at 50 µM). α-pinene and β-pinene demonstrated a significant neuroprotective effect against amyloid β exposure. α-pinene, β-pinene, terpineol, terpinolene and friedelin were associated with a variable inhibition of Aβ1-42 fibril and aggregate density.

CONCLUSIONS: The outcomes of this study underline a neuroprotective role of α-pinene and β-pinene against Aβ-mediated neurotoxicity associated with an inhibition of Aβ1-42 fibrilization and density. This demonstrates the bioactive potential of selected terpenes for consideration in the development of medicinal cannabis formulations targeting neurodegenerative diseases.