Characterization of the Chemopreventive Properties of Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy

Abstract

Hemp bioproducts hold great promise as valuable materials for nutraceutical and pharmaceutical applications due to their diverse bioactive compounds and potential health benefits. In line with this interest and in an attempt to valorize the Lazio Region crops, this present study investigated chemically characterized hydroalcoholic and organic extracts, obtained from the inflorescences of locally cultivated Felina 32, USO 31, Ferimon and Fedora 17 hemp varieties. In order to highlight the possible chemopreventive power of the tested samples, a bioactivity screening was performed, which included studying the antimutagenic activity, radical scavenging power, cytotoxicity in human hepatoma HepG2 cells, leakage of lactate dehydrogenase (LDH) and modulation of the oxidative stress parameters and glucose-6-phosphate dehydrogenase (G6PDH) involved in the regulation of the cell transformation and cancer proliferation. Tolerability studies in noncancerous H69 cholangiocytes were performed, too. The organic extracts showed moderate to strong antimutagenic activities and a marked cytotoxicity in the HepG2 cells, associated with an increased oxidative stress and LDH release, and to a G6PDH modulation. The hydroalcoholic extracts mainly exhibited radical scavenging properties with weak or null activities in the other assays. The extracts were usually well-tolerated in H69 cells, except for the highest concentrations which impaired cell viability, likely due to an increased oxidative stress. The obtained results suggest a possibility in the inflorescences from the Felina 32, USO 31, Ferimon and Fedora 17 hemp varieties as source of bioactive compounds endowed with genoprotective and chemopreventive properties that could be harnessed as preventive or adjuvant healing strategies.

Keywords: antimutagenic activity; cytotoxicity; entourage effects; glucose-6-phosphate dehydrogenase; hemp by-products; inflorescences; lactate dehydrogenase; oxidative stress; terpenoids.

Figure 7

Inhibition of glucose-6-phosphate dehydrogenase (G6PDH) activity by the organic extracts from (A) C. sativa var. Felina 32, (B) C. sativa var. USO31, (C) C. sativa var. Ferimon, and (D) C. sativa var. Fedora in HepG2 cells. The data are mean ± SE from at least three independent experiments (n = 3). ** p < 0.01 and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 9

Effects of the positive control quercetin (50 µM) and the organic extracts of Felina 32, USO31 and Fedora from the September harvest and Ferimon from the June harvest (50 µg/mL) on the GSH/GSSG ratio in HepG2 cells. GSH and GSSH were evaluated in cell lysates and calculated with respect to the calibration curves of GSH and GSSG. The data are mean ± SE from at least three independent experiments (n = 3). *** p < 0.001 vs. tBOOH (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 1

Inhibition of the tert-butyl hydroperoxide (tBOOH) mutagenicity (40 μg/mL) by hemp extracts obtained from the Monoecious Cultivars inflorescences collected in June and September in the WP2uvrAR strain. Values represent the mean ± SEM (n = 6). (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora.

Figure 2

Scavenger activity of the hemp extracts obtained from the Monoecious Cultivars inflorescences collected in June and September against the DPPH radical. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The values represent the mean ± SEM (n = 6).

Figure 3

Scavenger activity of hemp extracts obtained from the Monoecious Cultivars inflorescences collected in June and September against the ABTS radical. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The values represent the mean ± SEM (n = 6).

Figure 4

Cytotoxicity of hydroalcoholic extracts obtained from the June- and September-harvested inflorescences of the different varieties of Cannabis sativa L in human hepatoma HepG2 cells determined by using the MTT assay after 24 h of exposure. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The data are displayed as mean ± SE of at least three independent experiments (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 5

Cytotoxicity of the organic extracts obtained from the June- and September-harvested inflorescences of the different varieties of Cannabis sativa L in human hepatoma HepG2 cells determined using MTT assay after 24 h of exposure. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The data are displayed as mean ± SE of at least three independent experiments (n = 3). ** p < 0.01 and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 6

Cytotoxicity of organic extracts obtained from the inflorescences of different varieties of Cannabis sativa L in human hepatoma HepG2 cells determined by LDH release assay after 24 h of exposure. (A) C. sativa var. Felina 32 organic extract from September harvesting; (B) C. sativa var. USO31 organic extract from September harvesting; (C) C. sativa var. Ferimon organic extract from June harvesting; (D) C. sativa var. Fedora organic extract from September harvesting. The data are displayed as mean ± SE of at least three independent experiments (n = 3). *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 8

Effects of the organic extracts from the hemp varieties and of the positive control tBOOH (500 μM) on the intracellular ROS levels in HepG2 cells (A) C. sativa var. Felina 32 organic extract from September harvesting; (B) C. sativa var. USO31 organic extract from the September harvest; (C) C. sativa var. Ferimon organic extract from the June harvest; (D) C. sativa var. Fedora organic extract from September harvesting. ROS levels are expressed as oxidation index with respect to the basal levels. The data are mean ± SE from at least three independent experiments (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 10

Cytotoxicity of the hydroalcoholic extracts obtained from the June- and September-harvested inflorescences of the different varieties of Cannabis sativa L in human H69 noncancerous cholangiocytes determined via MTT assay after 24 h of exposure. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The data are displayed as mean ± SE of at least three independent experiments (n = 3). *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 11

Effects of the hydroalcoholic extracts from the hemp varieties on the intracellular ROS levels in human H69 noncancerous cholangiocytes after 24 h of exposure. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The data are displayed as mean ± SE of at least three independent experiments (n = 3).

Figure 12

Cytotoxicity of the organic extracts obtained from the June- and September-harvested inflorescences of the different varieties of Cannabis sativa L in human H69 noncancerous cholangiocytes determined via MTT assay after 24 h of exposure. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The data are displayed as mean ± SE of at least three independent experiments (n = 3). *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 13

Effects of the organic extracts from hemp varieties on the intracellular ROS levels in human H69 noncancerous cholangiocytes after 24 h of exposure. (A) C. sativa var. Felina 32; (B) C. sativa var. USO31; (C) C. sativa var. Ferimon; (D) C. sativa var. Fedora. The data are displayed as mean ± SE of at least three independent experiments (n = 3). ** p < 0.01, and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).