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

In this study, essential oils (EOs) and hydrolates (Hys) from Italian hemp (Cannabis sativa L. Kompolti cv.) and hop (Humulus Lupulus L., Chinook cv.) supply chains were chemically characterized and tested to investigate their apoptotic potential for the first time. Headspace–Gas Chromatography–Mass Spectrometry (HS-GC-MS) techniques were performed to describe their volatile chemical profile, highlighting a composition rich in terpene derivatives such as monoterpenes and sesquiterpenes among which β-myrcene, limonene, β-caryophyllene and α-humulene were the main constituents of EOs; in contrast, linalool, cis-p-menth-2,8-dien-1-ol, terpinen-4-ol, α-terpineol, caryophyllene oxide, and τ-cadinol were found in the Hys. The cytotoxicity activity on human leukemia cells (HL60), human neuroblastoma cells (SH-SY5Y), human metastatic adenocarcinoma breast cells (MCF7), human adenocarcinoma breast cells (MDA), and normal breast epithelial cell (MCF10A) for the EOs and Hys was studied by MTT assay and cytofluorimetric analysis and scanning and transmission electron microscopy were performed to define ultrastructural changes and the mechanism of cells death for HL 60 cells. An induction of the apoptotic mechanism was evidenced for hemp and hop EOs after treatment with the corresponding EC50 dose. In addition, TEM and SEM investigations revealed typical characteristics induced by the apoptotic pathway. Therefore, thanks to the integration of the applied methodologies with the used techniques, this work provides an overview on the metabolomic profile and the apoptotic potential of hemp and hop EOs and, for the first time, also of Hys. The findings of this preliminary study confirm that the EOs and Hys from Cannabis and Humulus species are sources of bioactive molecules with multiple biological effects yet to be explored.

As widely reported in the literature, the Cannabis genus has long been debated over the taxonomic rank of various groups and whether it is monospecific or polyspecific; it is currently believed to consist of a single species, namely Cannabis sativa L. [3] with subspecies each further subdivided into domestic and wild varieties [4]. C. sativa is a dioecious, rarely 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.

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).

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 (EC 50 ). The HL60 cells were shown to be the most sensitive to the treatments, with EC 50 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 EC 50 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).    Tables 3 and 4, the EC 50 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).  As reported in Tables 3 and 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).

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

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 EC 50 value for 24 h and live (double negative), early apoptotic (EA, AnnexinV-positive/PInegative 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.
Treatments with Hys of both plants did not determine an increase of the number of EA and LA cells (data not shown).

Ultrastructural Investigations by Transmission and Scanning Electron Microscopy
To define the effects of treatments with Kompolti hemp and Chinook hop EOs, SEM and TEM investigations were conducted on HL60 cells treated for 4 h, as well as on untreated and puromycin-treated cells, the latter as a hallmark of apoptosis.
In Figure 6A, the untreated cells show their typical morphology with a round shape and a cell membrane characterized by the presence of numerous regularly distributed cell protrusions. TEM analysis of untreated HL60 cells revealed its typical promyelocytic ultrastructure in which the nucleus occupies most of the cell volume and the cytoplasm is organized with the characteristic cellular compartments ( Figure 6B). The puromycin-treated cells showed the morphological features induced by the apoptotic mechanism, such as shrinkage of the cell, a smoother surface with round-shaped structures due to an intense surface blebbing ( Figure 6C,E). Dead cells were also observed ( Figure 6C).  By TEM analysis, puromycin treatment resulted in changes in cell ultrastructure, such as cytoplasmic and nuclear condensation, nuclear fragmentation (karyorrhexis), presence of multilamellar bodies, and cellular blebs. The pyknotic nucleus appeared like a half-moon, a typical feature of an apoptotic cell ( Figure 6D,F). Apoptotic characters were clearly found in the cells treated with hemp ( Figure 7A-D) and hop EOs ( Figure 7E-H). In hemp EO treatment, in addition to live cells with a round shape and a regular surface, cells with apoptotic blebs and swelling were found ( Figure 7A,C).  TEM analysis revealed a typical apoptotic ultrastructure and the presence of fragmentation of the cytoplasm and nucleus, inclusions and multilamellar bodies surrounded by cell membranes; cell blebs were clearly evident ( Figure 7B,D). Likewise, hop EO determined a cell volume shrinkage and a cellular blebbing ( Figure 7E-H). Pycnotic and fragmented nucleus and condensed cytoplasm were observed.

Discussion
The chemical composition of hemp and hop EOs and Hys was obtained by application of HS-GC-MS techniques. The obtained results highlighted a composition rich in sesquiterpenes for both EOs.
EO from C. sativa is a complex mixture of volatile compounds made up of more than 100 terpene derivatives [25]. Regarding Kompolti variety EO composition, in our study, limonene and β-caryophyllene were the most abundant components as also confirmed by other authors [26,27]. Compared to those conducted on the Kompolti variety, a greater number of reports concerning studies on other hop varieties such as Futura 75, Carmagnola, and Uso 31, whose compositions are characterized by an abundance of sesquiterpenes [28,29], are present. Nissen et al. [30] also investigated Carmagnola and Futura varieties but in contrast, they found a composition rich in monoterpenes with β-myrcene as the most abundant constituent in both EOs. Some studies attributed the variability of the hemp EOs chemical composition and its quality to more factors, such as the distillation technique and extraction time, the processing of plant material, the environmental factors, and cultivar type [27,[31][32][33][34].
Moreover, hop EO has a complex composition with a large number of volatile compounds belonging to different chemical classes that produce the distinct waves of scent for that type of hops according to their concentrations and interactions [35,36]. In particular, a recent review provides a number of insights into the various factors that can influence the presence of the volatiles in the hop EO which contribute to defining the aroma and flavor profile of beer [37]. Previous studies conducted on EOs obtained from wild hops grown in eastern Lithuania [38,39] revealed a complex composition by identifying up to 120 compounds among which α-humulene (11.1-33.4%), β-myrcene (15.7-21.1%), and γ-elemene (14%) were the most representative components in the different hop varieties investigated. In general, the monoterpene compounds such as β-myrcene and sesquiterpenes including α-humulene and β-caryophyllene are the most relevant compounds of hop EOs obtained from cones [40,41]. α-Humulene (4.87-15.26%) and τ-cadinol (6.83-28.06%) were found to be the major components in five varieties investigated by Kowalaska and colleagues [42]. However, the diversity of composition between the different hop EOs, as with other EOs, depends on the varietal characteristics [43,44] and several intrinsic and extrinsic factors. Among these, the geographical origin, the different cultivars, the climatic and soil conditions, and the variability of the secondary metabolism can be decisive in defining the profiling [39,45,46]. The differences are also due to the presence of parasites [47,48] and the use of pesticides, fungicides [49], and fertilizers whose presence also may affect the EO yield [47]. Equally important are the extraction methods used to isolate the EO [50,51], the method of storage and packaging after harvesting, and the temperature applied for drying which must not be too high to avoid oxidative degradation [52].
In our study, the evaluation of the EOs and Hys cytotoxic activity was performed against several cancer cell lines, i.e., human acute promyelocytic leukemia cells, human neuroblastoma cells, human metastatic mammary adenocarcinoma cells, human mammary adenocarcinoma cells, and a normal mammary epithelial cell, by means of the cytotoxic MTT assay. For the EOs, cytofluorimetric analysis and TEM and SEM investigations were also performed.
The most sensitive cell line to the EOs treatments was the HL60 leukemia cell line which showed an EC 50  was also obtained for the other studied cancer cell lines (EC 50 from a minimum of 237.20 to a maximum of 317.51 × 10 −3 mg/mL). The hemp EO was highly selective for the HL60 cell line compared to the normal MCF-10A cells with the SI value > 10 (SI = 20.11). Although the hop EO showed a low EC 50 value for HL60 cell line, it was not considered selective being cytotoxic for the normal cell line (SI = 7.57).
The Hys of hemp and hop were less active than the corresponding EOs. However, their cytotoxic activity was interesting in comparison to the Hys biological activity of other plant species [53,54] as the EC 50 values were rather low (EC 50 values from 4.49 to 326.76 mg/mL). Furthermore, the SI values for HL60 and SH-SY5Y were above 10 and thus both Hys could be considered selective. In contrast, for the adenocarcinoma cell lines, the SI values of both EOs were below 10 and they were not considered selective.
The cytotoxic activity of extracts and EOs from different varieties of C. sativa leaves, inflorescences, and seeds were observed on different cancer and normal cell lines [55][56][57][58][59][60]. Only one recent paper reported the cytotoxic activity of the Kompolti hemp EO variety on more human cancerous cell lines showing significant effects although with a lower tolerability in noncancerous cells [61].
To determine the type of cell death after EO treatment, HL60 cells, for which EC 50 values were very low, were further studied. By means of cytofluorimetric analysis, an induction of the apoptotic mechanism was evidenced with an apoptotic rate of 56.32% for hemp EO and 50.47% for hop EO after treatment with the corresponding EC 50 dose. In addition, TEM and SEM investigations were performed and comparison with puromycintreated cells revealed typical characteristics induced by the apoptotic pathway. Both the EOs determined an apoptotic morphological change such as extensive condensation of the chromatin, cytoplasmic shrinkage, blebbing of the plasma membrane, and the formation of apoptotic bodies [62].
In our investigation, limonene and β-caryophyllene were found to be the most abundant constituents of the Kompolti hemp EO and they have been shown to exert a chemopreventive activity and limit tumor growth and angiogenesis and induce apoptosis in several cancer cell models [63][64][65]. Furthermore, α-humulene, the most abundant component in Chinook hop EO, has also been reported to have anticancer and apoptotic inducing activities [66].
On the other hand, to the best of our knowledge, the apoptotic induction activity of H. lupulus EO had not been investigated until now. Finally, in the scientific literature, the description of the phytochemical composition of the hop and/or hemp Hys is missing. Therefore, in this study, in addition to evaluating the volatile profile of EOs, an investigation on the metabolomic profile of the respective hydrolates was carried out. The latter, performed by HS-GC-MS technique, represents the novelty of this work as well as the evaluation of the cytotoxic activity on both extractive products, also obtaining very promising results.

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.

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.

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.

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%.

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.

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 C 8 -C 30 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.

Cell culture and Maintenance
To 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% CO 2 condition. All experiments were performed with the cells in their logarithmic growth phase.

Cytotoxicity Assay
The cytotoxicity assay was performed using the MTT assay as described by Garzoli et al. [69]. The cells were seeded in 96 flat-bottomed tissue culture plate at seeding concentrations of 2 × 10 5 cells/mL for HL60 and SH-SY5Y cells, and 1 × 10 5 cells/mL for the MCF7, MDA, and MCF10A cells. The plates were incubated in a humidified incubator with 5% CO 2 at 37 • C for 24 h to allow the cells to attach to the bottom of the wells. A stock solution of EOs and DMSO at 50% was prepared to improve the solubility and bioavailability of essential oils. Subsequently, 10 two-fold different concentrations were added to the media, from 4 to 0.008 mg/mL for the EOs and from 500 to 1 mg/mL for the Hys. DMSO and H 2 O were used as solvent controls. Vinblastine was used as positive control. A dose-dependent treatment for 48 h was carried out to evaluate the cytotoxicity. Following each treatment, the media containing the compounds were aspirated and 100 µL of 0.5 mg/mL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well. After 3 h, the MTT solution was removed and 100 µL of DMSO was added to solubilize the purple-colored formazan product. The absorbance was read at 595 nm by a Tecan Sunrise™ UV-Vis spectrophotometer. The absorbance reading was corrected to background absorbance, and average reading was taken from three independent experiments. The cell viability expressed as percentage was calculated as follows: Cell viability % = Average absorbance of treated cells Average absorbance of untreated cells (control) × 100 The results were analyzed using GraphPad Prism version 8.0.2 for Windows (Graph-Pad Software, La Jolla, CA, USA). The half-maximal concentration (EC 50 ) for each biological replicate was determined using log-response curve. Overall, EC 50 was calculated from the average EC 50 of three independent biological replicates.

Selective Index
The Selective Index (SI) is defined as the selectivity of a tested substance against cancerous cells and a value higher than 10 belongs to a selected potential sample that can be further investigated [70].
It was calculated as follows: Selective index of a tumor cell line = EC50 of MCF10A cell line EC50 of a tumor cells line × 100

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 × 10 5 cells/mL on a six-well plate. After 24 h incubation, the cells were treated with EOs at the EC 50 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 × 10 4 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.

Scanning and Transmission Electron Microscopy
For scanning and transmission electron microscopy investigations, HL60 cells were seeded at a density of 1 × 10 6 cells/mL in a 10 mm petri dish and incubated for 24 h under appropriate conditions before adding treatments with hemp and hop EOs (EC 50 values). After 24 h of treatment, the cells were fixed with 4% paraformaldehyde and 5% glutaraldehyde (pH 7.2) in 0.1 M cacodylate buffer for 1 h at 4 • C (Karnowsky) and then post-fixed in 1% osmium tetroxide in cacodylate buffer for 1 h at 4 • C. A stepwise ethanol series was used to dehydrate the samples.
For scanning electron microscopy (SEM), cells were dried by the critical point method using CO 2 in a Balzers Union CPD 020 and were coated with gold in a Balzers MED 010 unit. Observations and micrographs were performed with a JEOL JSM 5200 electron mi-croscope (Jeol Ltd., Tokyo, Japan).
For transmission electron microscopy (TEM), the dehydrate samples were embedded in an Epon mix resin. Thin sections (50-70 nm) were obtained with Reichert Ultracut (Leica Mi-crosystems, Wetzlar, Germany) and LKB Nova ultramicrotome (LKB Vertriebs GmbH, Vienna, Austria) using a diamond knife, collected on copper grids, stained with uranyl acetate and lead citrate, and observed with a JEOL 1200EX II electron microscope (Jeol Ltd., Tokyo, Japan).

Conclusions
This paper aims to enrich the current state of knowledge on the potential of EOs and Hys from two singular industrial crops such as hop and hemp which have so far been less investigated than others.
Chemical analyses highlighted a rich volatile composition and the obtained results from performed biological assays showed the cytotoxic properties, on multiple human cell lines, of both Kompolti and Chinook EOs and, unexpectedly, also of the respective Hys. In particular, an induction of the apoptotic mechanism was evidenced for HL60 cells; in fact, TEM and SEM investigations revealed typical features induced by the apoptotic pathway. Certainly, this information will be useful increasing scientific the interest in Hys as a natural source of bioactive molecules. Future studies involving assays on pure compounds will be also conducted with the aim to establish a closer correlation between chemical composition and activity. Furthermore, further investigations will be needed to better understand the applications and uses of these EOs and Hys in various fields such as pharmaceutical ones.