A Nrf-2 Stimulatory Hydroxylated Cannabidiol Derivative from Hemp (Cannabis sativa)

Abstract

A phytochemical analysis of mother liquors obtained from crystallization of CBD from hemp (Cannabis sativa), guided by LC-MS/MS and molecular networking profiling and completed by isolation and NMR-based characterization of constituents, resulted in the identification of 13 phytocannabinoids. Among them, anhydrocannabimovone (5), isolated for the first time as a natural product, and three new hydroxylated CBD analogues (1,2-dihydroxycannabidiol, 6, 3,4-dehydro-1,2-dihydroxycannabidiol, 7, and hexocannabitriol, 8) were obtained. Hexocannabitriol (8) potently modulated, in a ROS-independent way, the Nrf2 pathway, outperforming all other cannabinoids obtained in this study and qualifying as a potential new chemopreventive chemotype against cancer and other degenerative diseases.

Figure 1

Structures of Δ⁹-THC (1), CBD (2), and cannabitwinol (3).

Figure 2

LC-MS chromatogram of the C. sativa mother liquors extract and (left) selected cannabinoid cluster from MS/MS-based molecular network (nodes are labeled with the parent m/z ratio; edge thickness is related to the cosine similarity score; colors: green for compounds isolated by RP-HPLC and fully assigned by HR-MSMS and NMR; white for the unassigned nodes; yellow ring for cannabidiol); (right) LC-MS/MS spectrum of cannabidiol (2).

Figure 3

Structures of selected phytocannabinoids obtained as constituents of the mother liquors obtained from the crystallization of CBD (2).

Figure 4

Diagnostic 2D NMR correlations detected for 6. (Left) COSY (red bolded) and key HMBC (arrows) correlations. (Right) Key NOESY (red arrows) correlations.

Figure 5

Effects of selected phytocannabinoids on Nrf2 activity. HaCaT-ARE-Luc cells (15 × 10⁴ cells/mL) were treated with 1–10–25 μM concentrations of each compound for 6 h. Luciferase activity was measured in the cell lysates, and the results are represented as percentage-fold induction relative to 20 μM tert-butyl-hydroquinone (TBHQ), taken as 100%.

Figure 6

Intracellular accumulation of ROS detected using 2′,7′-dihydrofluorescein-diacetate (DCFH-DA) in HaCaT cells. tert-Butyl-hydroperoxide (TBHP) (0.4 mM) was used as a standard (100%) ROS producer, while N-acetylcysteine (NAC) (15 mM) was used as a positive control that inhibited TBHP-induced ROS production. Cells were treated with increasing concentrations (1–10–25 μM) of hexocannabitriol (8).