New Cannabinoids and Chlorin-Type Metabolites from the Flowers of Cannabis sativa L.: A Study on Their Neuroblastoma Activity

Abstract

Background/Objectives: Cannabis sativa has been utilized for medical purposes for thousands of years. It continues to be recognized as a plant with an extensive variety of medicinal and nutraceutical uses today. In this study, a chemical investigation of the flowers of C. sativa isolated by using a variety of chromatographic techniques led to the isolation of eleven compounds. These purified compounds were evaluated for antitumor activity against SK-N-SH neuroblastoma cells. Methods: The compounds were isolated by using chromatographic techniques. Their structures were identified by the examination of spectroscopic methods, including 1D (¹H, ¹³C, and DEPT) and 2D (COSY, HSQC, HMBC, and NOESY) nuclear magnetic resonance (NMR) spectra and mass spectrum, together with the comparison to those reported previously in the literature. The evaluation of toxicity on SK-N-SH cells was performed by the MTT method. Results: Eleven compounds were isolated from the flowers of C. sativa, including two new compounds, namely cannabielsoxa (1), 13²-hydroxypheophorbide c ethyl ester (2), and six known cannabinoids (6-11), together with the first isolation of chlorin-type compounds: pyropheophorbide A (3), 13²-hydroxypheophorbide b ethyl ester (4), and ligulariaphytin A (5) from this plant. The results also demonstrated that cannabinoid compounds had stronger inhibitory effects on neuroblastoma cells than chlorin-type compounds. Conclusions: The evaluation of the biological activities of compounds showed that compounds 4-10 could be considered as the potential compounds for antitumor effects against neuroblastomas. This is also highlighted by using docking analysis. Additionally, the results of this study also suggest that these compounds have the potential to be developed into antineuroblastoma products.

Keywords: Cannabis sativa L.; SK-N-SH cells; cannabinoid; chlorin-type compound.

Figure 1

The structure of isolated compounds.

Figure 2

Key COSY () and HMBC () correlations of 1 and 2.

Figure 3

Key NOESY correlations of 1.

Figure 4

Antitumor effects of compounds from C. sativa on SK-N-SH neuroblastoma cells. SK-N-SH cells were seeded into 96-well plates at a density of 5 × 10⁴ cells/mL and treated with the concentration of the compounds at 10 μM for 24 h. Cell viability was determined using the MTT assay, as described in the ‘Methods’ section. Data represent the means ± standard deviation (S.D.) (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test, and differences were statistically significant at *** p < 0.001 compared to DMSO-treated controls.

Figure 5

Antitumor effects of compounds on SK-N-SH neuroblastoma cells. SK-N-SH cells were seeded into 96-well plates at a density of 5 × 10⁴ cells/mL and treated with compounds at concentrations of 2.5, 5, and 10 μM for 24 h. Cell viability was assessed using the MTT assay, as described in the ‘Methods’ section. Data represent the means ± standard deviation (S.D.) (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test, and differences were statistically significant at * p < 0.05 and *** p < 0.001 compared to DMSO-treated controls.

Figure 6

IC₅₀ values of compounds 6–10.

Figure 7

Molecular docking simulation of compounds 6–8 and the positive control with the human neuroblastoma SK-N-SH protein (PDB ID: 4KUM). (A,B) Two-dimensional and three-dimensional binding interactions of compound 6 with 4KUM. (C–F) Structural and interaction analyses of compounds 7 and 8, respectively. (G,H) Binding interactions of the positive control with 4KUM. Compounds 6–8 have transparent surfaces on their ligand-binding pockets (left panels). The right panels depict interactions between the ligands and the protein’s amino acid residues, such as hydrogen bonds (pink) and pi–pi stacking interactions (green).

Scheme 1

Extraction and isolation for compounds 1–11.