Chromatographic Analyses, In Vitro Biological Activities, and Cytotoxicity of Cannabis sativa L. Essential Oil: A Multidisciplinary Study

Abstract

Due to renewed interest in the cultivation and production of Italian Cannabis sativa L., we proposed a multi-methodological approach to explore chemically and biologically both the essential oil and the aromatic water of this plant. We reported the chemical composition in terms of cannabinoid content, volatile component, phenolic and flavonoid pattern, and color characteristics. Then, we demonstrated the ethnopharmacological relevance of this plant cultivated in Italy as a source of antioxidant compounds toward a large panel of enzymes (pancreatic lipase, α-amylase, α-glucosidase, and cholinesterases) and selected clinically relevant, multidrug-sensible, and multidrug-resistant microbial strains (Staphylococcus aureus, Helicobacter pylori, Candida, and Malassezia spp.), evaluating the cytotoxic effects against normal and malignant cell lines. Preliminary in vivo cytotoxicity was also performed on Galleria mellonella larvae. The results corroborate the use of this natural product as a rich source of important biologically active molecules with particular emphasis on the role exerted by naringenin, one of the most important secondary metabolites.

Keywords: Cannabis sativa L.; Galleria mellonella; antimicrobial activity; antioxidant activity; biofilm; cancer cell cytotoxicity; chromatographic analysis; essential oil; naringenin.

Figure 1

Reflectance curves of hemp essential oil (EO; gray) and its aromatic water (yellow).

Figure 2

The evaluation of minimum inhibitory concentration (MIC) of the hemp EO versus Staphylococcus aureus determined using the alamarBlue^® (AB) assay. (A) Representative image of colorimetric MIC determination using AB at 24 h of incubation. The white rectangle indicates the MICs at 8 mg/mL S. aureus 101. (B) The plot shows the percentage reduction of AB in the S. aureus broth cultures at different hemp EO concentrations compared to the corresponding untreated samples (0) evaluated as indicated in the experimental section of this manuscript. MHB: Mueller–Hinton broth; EtOH: ethanol. Data are presented as the mean of three replicates of three independent experiments.

Figure 3

The evaluation of minimum biofilm eradication concentration (MBEC) of the hemp EO versus Staphylococcus aureus biofilm determined using the AB assay, live/dead staining, and fluorescent microscopy analysis and colony-forming unit (CFU) counts. (A) Representative image of colorimetric MBEC determination using AB. The white rectangles indicate the MBECs at 24 mg/mL S. aureus 105. (B) The plot shows the percent reduction of AB in the S. aureus broth cultures at different hemp EO concentrations compared to the corresponding untreated samples (0) evaluated as indicated in Section 2. (C) CFU count of treated and untreated S. aureus biofilms. (D) Representative image of S. aureus (a) untreated and (b) treated biofilms with 24 mg/mL hemp EO. The biofilms were stained with a live/dead kit and visualized using fluorescent microscopy. The green fluorescence indicates live cells, whereas the red fluorescence indicates dead cells or cells with a compromised cell wall. TSB: tryptic soy broth; GLU: glucose. Data are presented as the mean of three replicates of three independent experiments; * p ˂ 0.05 vs. the controls (0).

Figure 4

Cytotoxic effect of hemp EO in different human cell lines after 24 h of exposure. MCF-7, estrogen-dependent breast cancer cells; MDA-MB-468, triple-negative breast cancer cells; Caco-2, colorectal adenocarcinoma cells; Mz-ChA-1, cholangiocarcinoma cells; H69, nonmalignant immortalized cholangiocytes. Data are the mean ± SEM from almost two independent experiments with three technical replicates (n = 6); * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control determined by ANOVA followed by Dunnett’s multiple comparison post hoc test.

Figure 5

Survival curves of Galleria mellonella larvae after hemp EO treatment. Each data point represents the percent survival of G. mellonella larvae (N = 10/group, repeated on three different occasions), following injection with test samples and incubation at 37 °C. Control larvae were not injected or injected with phosphate-buffered saline (PBS) plus Tween 20 to simulate the trauma associated with the administration. Larvae were monitored for 24 h up to five days.