Hemp (Cannabis sativa L., Kompolti cv.) and Hop (Humulus lupulus L., Chinook cv.) Essential Oil and Hydrolate: HS-GC-MS Chemical Investigation and Apoptotic Activity Evaluation

2.2. Chemical Analyses of Chinook Hop and Kompolti Hemp Hys and EOs

The chemical composition of the EOs was obtained by GC/MS technique. In total 53 compounds were identified of which 28 were in hop EO and 31 in hemp EO (Table 1). In hop EO, the sesquiterpenes (79.7%) prevailed over the monoterpenes contrary to the hemp EO, where the monoterpene fraction was more abundant. Qualitative and quantitative differences were found between the two EOs. In detail, humulene (38.0%) followed by β-caryophyllene (12.2%), δ-cadinene (9.4%), and τ-cadinol (4.5%) were the major sesquiterpenes detected in hop EO. In contrast, in hemp EO, β-caryophyllene (22.2%) prevailed over humulene (6.5%). Further, among monoterpenes, limonene (27.9%), β-myrcene (12.4%), α-pinene (8.6%), and terpinolene (6.2%) were the principal component in hemp EO while β-myrcene (9.4%) was the main one in hop EO.

A series of minor compounds have only been detected in one oil or another. Among them, some terpenes with a percentage value greater than 1% such as linalool (1.3%), (Z)-methyl-geranate (1.2%), α-copaene (1.8%), γ-muurolene (3.6%), α-selinene (2.1%), and α-eudesmol (1.4%) were characteristic of the hop EO chemical profile while cis–β-ocimene (2.1%) and β-maaliene (2.3%) were found in hemp EO. The chromatograms of both EOs were reported in Figure 1.

The volatile composition of hop and hemp Hy carried out by HS-GC-MS technique highlighted the presence of 29 compounds, 20 in the hop Hy and 14 in that of hemp (Table 2). The chemical profile of the two Hys differed in that τ-cadinol (22.9%) followed by linalool (21.2%) and α-terpineol (12.7%) were the most abundant components of hop Hy, while α-terpineol (31.2%), cis–p-mentha-2,8-dien-1-ol (25.7%), and terpinen-4-ol (18.1%) were the ones in hemp Hy. Only five components, namely hexanal, linalool, isoborneol, terpinen-4-ol, and α-terpineol were detected in both Hys; moreover, sesquiterpenes such as caryophyllene oxide, τ-cadinol, germacrene D, and α-eudesmol, with percentage values ranging from 1.2 to 22.9%, were characteristic only of the hop Hy. In general, the monoterpene content exceeded the sesquiterpene one. The chromatograms of both Hys are reported in Figure 2.

2.3. Cytotoxicity Determination of EOs and Hys Using MTT Assay

The cytotoxic activity of hemp and hop EOs and Hys against different human cells lines was examined by dose-dependent approach at 48 h of treatments. In Table 3, we reported the concentrations of EOs and Hys able to reduce the 50% of cell viability (EC₅₀). The HL60 cells were shown to be the most sensitive to the treatments, with EC₅₀ values of 10.49 × 10⁻³ mg/mL and 38.92 × 10⁻³ mg/mL after 48 h of hemp and hop EOs incubation, respectivetly. The hemp EO was more active toward the neuroblastoma cells (SH-SY5Y), with an EC₅₀ value of 95.87 × 10⁻³ mg/mL, than that of hop which had a value of 315.60 × 10⁻³ mg/mL. Both EOs were also quite active against the two breast cancer cells, MCF7 and MDA (241.10 × 10⁻³ mg/mL and 230.17 × 10⁻³ mg/mL for hemp EO, 237.20 × 10⁻³ mg/mL and 317.51 × 10⁻³ mg/mL or hop EO, respectively).

Concerning hemp and hop Hys, EC₅₀ values were 6.05 mg/mL and 4.90 mg/mL, respectively. Likewise, low EC₅₀ values were obatined for the treatment of SH-SY5Y cells (4.90 mg/mL for hemp and 10.46 mg/mL for hop). Higher EC₅₀ values for the corresponding treatment on MCF-7 and MDA cells were obtained (Table 4). Figure 2 shows the bar graphs of the EC₅₀ values of EOs (Figure 3a) and Hys (Figure 3b) obtained from Kompolti hemp and Chinook hop.

As reported in Table 3 and Table 4, the EC₅₀ values obtained from the treatment against the normal human MCF10A cell line were used to define the Selectivity Index (SI). For EOs (Table 3), the lowest SI value was observed for human leukemic cells (20.11 × 10⁻³ mg/mL for C. sativa EO and 7.57 × 10⁻³ mg/mL for H. lupulus EO). In the case of Hys, the lowest SI values were observed for C. sativa (37.25 mg/mL and 45.99 mg/mL) and for H. lupulus (15.67 mg/mL and 36.53 mg/mL), against leukemia and neuroblastoma cells, respectively (Table 4).

2.4. Flow Cytometry Analysis

To assess the cell death mechanism occurred by the EOs treatments, the reduced cell viability of HL60 cells was further investigated by Annexin/FITC and propidium iodide in flow cytometry analysis. Each treatment was carried out using the corresponding EC₅₀ value for 24 h and live (double negative), early apoptotic (EA, AnnexinV-positive/PI-negative staining), late apoptotic (LA, double positive), and necrotic cells (PI positive) per-centages were defined. The apoptotic rates, resulting from the sum of the percentages of AnnexinV-positive/PI-negative and double-positive stained cells, are shown in Figure 4.

In ctrl (control) cells, the apoptotic rate was 7.87% ± 1.68% whereas in puromycin treated cells apoptotic cells was 78.05% ± 3.47%. After 24 h of treatment with hemp and hop EOs, a high level of Annexin V-positive/PI-negative staining and double-positive staining were recorded (56.32% ± 6.78% and 50.47% ± 5.90%, respectively) (Figure 5).

Treatments with Hys of both plants did not determine an increase of the number of EA and LA cells (data not shown).

4.2. Plant Material

Hops subjected to this study were the Chinook variety, grown in San Nicandro Garganico (Foggia, Italy), by the Agricola Vocino Farm in 2021.

The choice of this variety of hop is linked to its high content of α-acids (greater than 10%), which allow the plant to resist extreme abiotic events such as heat and drought typical of the growing area, but also biotic such as attacks from the downy mildew “Fusarium canker”.

The plants were positioned with about 1 m between one another, and between the individual rows, the distance maintained was about 3 m. Therefore, the density per hectare was about 3000 plants for a total of 150 plants in three rows. Irrigation was carried out by drop without resorting to any treatment or chemical fertilization while the tillage was mostly manual. The manual harvesting of the plants was carried out towards the end of August; over a period of 15–20 days, at least 2/3 collections were carried out. The drying of the hop cones was performed in a natural way, inside a dark room in order to protect hop from the sun’s rays, ventilated (in a natural or artificial way), and possibly with humidity control (about 50% RH). After about 5 days, the hops were vacuum-packed and stored in a cool and dry place. In these conditions, the hop inflorescences are usable for about 24 months.

The Kompolti hemp variety was grown in Torre del Lago, Lucca, Italy, by the Carmazzi Farm in 2019. This hemp variety is of Hungarian origin and is characterized by a low/no THC content and medium/low CBD content (THC: <0.2% CBD: 2.0–10.0%).

The sowing was done in April in a thermo-controlled greenhouse and under LED lamps. The plants were arranged at about 1.5 m from each other, maintaining a distance of 4 m between the individual rows. Subsequently, the seedlings were transplanted into 8 L pots and kept in the greenhouse. At the beginning of June, when the plant developed a suitable root system, it was transplanted in the open field. The soil was set aside during the winter and prepared with a superficial mechanical processing so as not to compromise the microbiome, which is important for the rooting of the plant itself.

The apical topping technique was applied to limit vegetative growth and to increase the production of apical and lateral inflorescences. Drip irrigation was performed throughout the vegetative phase and mulched with biodegradable corn cloth without resorting to any phytochemical treatment. The collection of the inflorescences, carried out early in the morning to preserve the aromatic component of the inflorescences, began at the end of August until the beginning of September and no presence of parasites was ever observed. After harvesting, the inflorescences were manually cleaned of the leaves and then placed in vacuum packs and stored in the freezer at a temperature of −20 °C.

4.3. Hydrodistillation (HD)

Chinook hop EO and Hy were obtained by hydrodistillation (HD). In particular, 1.4 kg of dried plant material were distilled in two round flasks of 10 L capacity, each containing 700 g of hop and 6 L of distilled water. Hop inflorescences were soaked in distilled water overnight, and then HD was performed for 4 h using a Clevenger-type apparatus and a heating mantle system Falc MA (Falc Instruments, Treviglio, Italy). Hop EO and hydrolates were collected separately and kept at 4 °C until further analysis. The EO yield accounted for 0.37% w/w.

4.4. Moisture Content Evaluation of Frozen Hemp

In order to determine the water content, 3 homogeneous samples of Kompolti frozen inflorescences were heated at 100 °C on a thermo balance (Scaltec SMO 01, Burladingen, Germany), and an average value of 60.6% was obtained.

4.5. Microwave-Assisted Extraction (MAE)

Kompolti hemp EO and Hy were obtained through the advanced microwave extraction system Milestone ETHOS X (Milestone, Italy). A total of 3.3 kg of frozen plant material was left to defrost for 30 min, and then subjected to 3 MAE runs. In each of them, 1.1 kg of inflorescences was processed with 1.3 L of distilled water, in a glass reactor of 12 L capacity, and placed within the microwave reactor, operating at 2.45 GHz. The microwave power was set at the maximum value delivered by the instrument, namely 1800 W, and the extraction time was 3 h. A stainless-steel Clevenger-type apparatus above the system, and a Chiller Smart H150-2100S by Labtech srl (Sorisole, Bergamo, Italy), which kept water temperature at 8 °C, enabled the distillation process. Kompolti EO and hydrolate were separated and stored in glass vials at 4 °C before analysis. The EO yield was estimated on dry matter (w/w) and was 0.35%.

4.6. Headspace–Gas Chromatography–Mass Spectrometry (HS-GC-MS)

The volatile profile of the EOs and HYs vapor phase was investigated using a Perkin-Elmer Headspace Turbomatrix 40 (Waltham, MA, USA) autosampler connected to GC-MS. The operative conditions were performed as previously described [67]. About 1 mL of each EO and 2 mL of each Hy were placed separately in 20 mL vials sealed with headspace PTFE-coated silicone rubber septa and caps. The identification and quantifications of compounds were performed as described in Section 4.7.

4.7. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

To characterize the chemical composition of all samples, a Clarus 500 model Perkin Elmer (Waltham, MA, USA) gas chromatograph coupled with a mass spectrometer and equipped with a FID (flame detector ionization) was used. Chromatographic separation was performed using a Varian Factor Four VF-1 capillary column and gas carrier was He at flow rate of 1.0 mL/min in constant flow mode. The GC operative conditions followed Ovidi et al. [68]. The volatile separated compounds were identified by matching their mass spectra with those stored in the Wiley 2.2 and Nist 02 mass spectra libraries database and by comparison of their linear retention indices (LRIs), relative to C₈–C₃₀ n-alkanes analyzed under the same conditions, with those available in the literature. Relative concentrations of individual compounds were expressed as percentage of the relative peak area to that of the total peak area without the use of an internal standard and any factor correction. All analyses were carried out in triplicate.

4.8. Cell culture and Maintenance

To evaluate the effect of EOs and HYs on cell viability, five different cell lines were used: human acute promyelocytic leukemia cells (HL60, ATCC^® CCL-240), human neuroblastoma cells (SH-SY5Y, ATCC^® CRL-2266), human metastatic adenocarcinoma breast cells (MCF7, ATCC^® HTB-22™), human adenocarcinoma breast cells (MDA, ATCC^® MB-231), and normal breast epithelial cell (MCF10A. ATCC^® CRL-10317™).

The HL60 were cultured in RPMI-1640 supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. The SH-SY5Y, MCF7, and MDA cells were cultured in DMEM F-12 supplemented with 10% FBS, 1% glutamine, and 1% penicillin-streptomycin. The MCF10A were maintained in DMEM F-12 medium supplemented with 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor (EGF), 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 5% Horse Serum (HS), 1% glutamine, and 1% penicillin-streptomycin; before being seeded for the viability assay, the culture medium was deprived of EGF and the HS was reduced to 2%. All the cell lines were maintained at 37 °C in a humidified 5% CO₂ condition. All experiments were performed with the cells in their logarithmic growth phase.

4.9. Flow Cytometry

To determine the apoptotic induction activity of the EOs of Kompolti hemp and Chinook hop, a flow cytometry analysis was performed on HL60 cells using the Annexin V-FITC and PI apoptosis kit (eBioscience., San Diego, CA, USA). HL60 cells were plated at a density of 5 × 10⁵ cells/mL on a six-well plate. After 24 h incubation, the cells were treated with EOs at the EC₅₀ concentration.

After 24 h, the cells were stained with an Annexin V-FITC conjugate and PI, and the emitted fluorescence was analyzed using a flow cytometer (BD Accuri C6 Plus, BD Italy S.p.A, Milano, Italy). A total of 1 × 10⁴ events per sample were acquired and the obtained “dot plot”, in which each point corresponds to a single event, was recorded. The experiments were repeated three times.

The authors are grateful to Agricola Vocino Farm, Italy, for providing hops material and Carmazzi Farm for providing hemp material.