Extraction, purification and in vitro assessment of the antioxidant and anti-inflammatory activity of policosanols from non-psychoactive Cannabis sativa L

Abstract

Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans-esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity. HPLC-ELSED analysis of the purified mixture from hemp wax revealed C₂₆OH and C₂₈OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes.

Keywords: Anti-inflammatory activity; Antioxidant activity; Cannabis sativa L.; Extraction; HPLC; Policosanols.

Graphical abstract

Fig. 1

HPLC-ELSD chromatogram of the PC mixture purified from hemp wax using flash chromatography (fractions 22–26). For peak identifications see Table 1.

Fig. 2

Quantification of ROS in HaCat cell line treated or not with PCs exposed to H₂O₂. Intracellular ROS levels were quantified after treatment with 350 μM H₂O₂ in HaCat cells pretreated or not with different concentrations of PCs. The ROS levels were measured from relative fluorescence intensity with H₂DCF-DA. Data are expressed as mean ± S.D. (n = 7); significance one-way ANOVA with Tuckey's multiple comparison test, ***p < 0.001 and **p < 0.01 vs. Ctrl, ⁺⁺p < 0.01 and+p < 0.05 vs H₂O₂).

Fig. 3

Effect of PCs on H₂O₂ induced intracellular ROS content in HaCaT cells. (A) HaCaT cells were pretreated with PCs at 100 μg/mL for 12 h before to exposure to 350 μM H₂O₂ for 30 min. ROS content was detected by CellROX-Green and examined with 20 × fluorescence microscope. Scale bar: 100 μm. (B) Fluorescence quantitative analysis by ImageJ. Data are expressed as mean ± S.D. (n = 7) and the significance one-way ANOVA with Tuckey's multiple comparison test p < 0.001 vs. Ctrl and ***p < 0.001 vs·H₂O₂ treated group.

Fig. 4

Effect of PCs on the MK2 and HO-1 protein expression in H₂O₂-treated HaCaT cells. (A) Representative Western blot of MK2 protein (49 kDa) and HO-1 (30 kDa) of HaCaT cell lysate pretreated with PCs at 100 μg/mL for 12 h prior to treatment with 350 μM of H₂O₂ for 30 min. The uncropped version of the Western blot is also available (Fig. S3, Supplementary Information). (B, C) Densitometric analyses of protein levels of MK2 and HO-1. Densitometry values were normalized to the protein loading control, GAPDH. The values are expressed as the mean ± SD of four independent experiments (n = 4 per group). Significance: one-way ANOVA with Tuckey's multiple comparison test ****p < 0.0001, ***p < 0.001, **p < 0.01 and * p < 0.05.

Fig. 5

Anti-inflammatory activity of PCs from hemp wax. Values are reported as fold decrease of luciferase signal with respect to cells treated with TNF-α. Dose-dependent activity of hemp wax in a concentration range between 5 and 100 μg/mL and the results from the one-way ANOVA analysis followed by multi-comparison test (****p < 0.0001, ***p < 0.001 and * p < 0.05).