Evaluation of two different Cannabis sativa L. extracts as antioxidant and neuroprotective agents

Abstract

Cannabis sativa L. is a plant that contains numerous chemically active compounds including cannabinoids such as trans-Δ-9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), and flavone derivatives, such as luteolin-7-O-glucuronide and apigenin glucuronide. In particular, the polar fraction of hemp including many phenolic compounds has been overlooked when compared with the more lipophilic fraction containing cannabinoids. Therefore, the aim of this study was to assess two extracts of industrial hemp (C. sativa) of different polarity (aqueous and hexane) by evaluating their antioxidant profile and their neuroprotective potential on pharmacological targets in the central nervous system (CNS). Several assays on in vitro antioxidant capacity (DPPH, superoxide radical, FRAP, ORAC), as well as inhibition of physiological enzymes such as acetylcholinesterase (AChE) and monoaminooxidase A (MAO-A) were carried out in order to find out how these extracts may be helpful to prevent neurodegenerative disorders. Neuro-2a cell line was selected to test the cytotoxic and neuroprotective potential of these extracts. Both extracts showed striking antioxidant capacity in the FRAP and ORAC assays, particularly the hexane extract, and interesting results for the DPPH and superoxide radical uptake assays, with the aqueous extract standing out especially in the latter. In enzyme inhibition assays, the aqueous extract showed AChE and MAO-A inhibitory activity, while the hexane extract only reached IC₅₀ value for AChE inhibitory bioassay. Neuro-2a assays demonstrated that polyphenolic extract was not cytotoxic and exhibited cytoprotective properties against hydrogen peroxide and antioxidant response decreasing reactive oxygen species (ROS) production. These extracts could be a source of compounds with potential benefit on human health, especially related to neurodegenerative disorders.

Keywords: antioxidant; cannabidiol (CBD); cannabinoid; central nervous system (CNS); enzymatic inhibition; oxidative stress; polyphenol; trans-Δ-9-tetrahydrocannabinol (Δ9-THC).

FIGURE 1

CNS enzyme inhibitions. IC₅₀ values were calculated by non-linear regression. (A) Acetylcholinesterase inhibition profiles of Cannabis extracts and galantamine. (B) Monoamine oxidase A (MAO-A) inhibition profiles of polyphenolic Cannabis extract and clorgiline.

FIGURE 2

Antioxidant profile. IC₅₀ were calculated by non-linear regression. (A) DPPH inhibition of hemp extracts. (B) Hemp extracts scavenge superoxide radicals generated by the xanthine/xanthine oxidase system. Ascorbic acid was used as reference antioxidant.

FIGURE 3

Mitochondrial response. (A) Cytotoxicity of Neuro-2a cells after exposure to different concentrations of Cannabis sativa aqueous extract. (B) Cytoprotective effects of Cannabis sativa aqueous extract versus hydrogen peroxide (300 µM). (C) Inverted microscope images of Neuro-2a cells (0–500 μg/ml) Note: **p < 0.01 versus H₂O₂; ****p < 0.001 versus H₂O₂; ####p < 0.001 versus control.

FIGURE 4

ROS production in Neuro-2a cells subjected to oxidative stress by hydrogen peroxide ((300 µM) and treatments with Cannabis sativa aqueous extract (25–200 μg/ml). Data are expressed as percentage over control cells and the assay was carried out for 90 min in order to measure intracellular ROS production. Note: ####p < 0.0001 versus control. Significant differences appeared at the starting point for H₂O₂-N2a cells over control cells. Since 0 min, 25 and 50 μg/ml pre-treatments were associated with significant differences (p < 0.0001). However, 100 and 200 μg/ml pre-treatments were associated with significant differences (p < 0.0005 and p < 0.05, respectively). From 70 min and above, highest significant differences were reached (p < 0.0001) for all treatments against hydrogen peroxide.