2.2. LVI-GC/MS Chemical Composition of C. sativa L. Hy

By LVI-GC/MS analyses, twelve components were detected and identified in Hy (Table 2). Among these, α-terpineol (14.5%) was the main detected monoterpene followed by linalool (3.2%) and fenchol (1.2%). The relatively more abundant compound was 2-methylisoborneol (19.2%), a non-canonical monoterpene, known to be responsible, together with geosmin, for the musty and earthy odor of water caused by various microorganisms [25]. This compound was not present in the EO, as well as other compounds such as limonene-1,2-diol (5.2%), 8-hydroxylinalool (4.2%), sobrerol acetate (9.6%) and sobrerol (6.4%). These latter two had already been detected in the Hy of the resin of Pistacia lentiscus [26]. In particular, sobrerol is a monoterpene with two hydroxyl functions known for its radical scavenging activity [27]. Limonene-1,2-diol is a monoterpenoid to which anti-inflammatory and antifungal activities have been attributed [28,29]. This bioactive compound, in addition to being naturally present in some plants [30], can also be produced by the biotransformation of limonene [30]. 8-Hydroxylinalool is a linalool-derived compound widely present in Vitis vinifera [31] and, as recently reported, in Camellia sinensis var. assamica ‘Hainan dayezhong’ [32]. This terpene oxide was demonstrated to have a special anti-microbial activity against Gram (±) bacteria strains and against pathogenic fungi [33].

No sesquiterpenes were detected. Other non-terpene components were identified with relevant average percentage values such as 4-OH-butanoic acid (10.0%) and 3-buten-1,2-diol-1(2-furanyl)-2-methyl (16.1%).

The presence of both low-polarity volatile compounds and low-volatility hydrophilic compounds makes complex the analysis of the Hy.

Most studies apply a liquid–liquid extraction with an organic solvent and subsequent introduction into a GC system. But, to the best of our knowledge, only one paper has reported the use of direct hydrosol analysis by GC-MS with encouraging results [34]. In our case, the use of a TOTAD (Through Oven Transfer Adsorption Desorption) interface allows high quantities (up to a few hundred microliters) of aqueous sample to be injected directly into a capillary gas chromatography column, overcoming the limitations of the injectable volume and providing the possibility of significantly increasing sensitivity if the Hys are very diluted.

2.3. DI-SPME/GC-MS Chemical Composition of C. sativa L. Hy

The sampling and analysis method applied to C. sativa Hy allowed the identification of twenty-two volatile components (Table 3). The monoterpene fraction (88.6%) was significantly higher than the sesquiterpene fraction (7.8%). According to the data obtained by LVI-GC/MS, α-terpineol (26.4%) was the principal monoterpene. It has countless biological activities including an anticancer effect [35]. Following this, linalool (23.2%) and fenchol (21.2%) were the compounds detected with a similar relative abundance. Among these oxygenated monoterpenes, it has been recently reported that linalool induces apoptosis of cancer cells via oxidative stress, and at the same time protects normal cells [36] while fenchol is known to possess antibacterial activity at a broad spectrum [37]. On the other hand, among the sesquiterpenes, β-caryophyllene, although with relatively low percentage values, was the most abundant. From the obtained data, it can be stated that DI-SPME/GC-MS compared to LVI/GC-MS, was the most adequate technique for the detection of the sesquiterpene component. On the other side, the LVI/GC-MS technique allowed the detection of other monoterpene compounds such as limonene-1,2-diol, 8-hydroxylinalool and sobrerol.

To the best of our knowledge, the present work is the first to have investigated the compositional profile of the Hy of C. sativa Carmagnola cv. In fact, in the scientific literature, one paper is available that describes the chemical composition of C. sativa Hy, but concerning the Kompolti variety [38].

3.2. LVI-GC-MS Analysis of C. sativa Hydrolate

Direct injection in a GC-MS system of large aqueous volume was performed using a Konik instrumentation equipped with a 5000C gas chromatograph coupled to a mass spectrometer detector MS Q2 (Sant Cugat del Vallés, Barcelona, Spain) and equipped with a TOTAD (Through Oven Transfer Adsorption Desorption) interface, operating as described in [46] with minor modifications. The adsorption temperature was kept at 60 °C for 6 min under a nitrogen gas flow of 150 mL/min to eliminate the solvent (water) present in the Hy sample (20 µL), which was transferred to the interface with a methanol flow of 0.1 mL/min for 1 min. The analytes were desorbed at 260 °C for 5 min.

The gas chromatograph operated with a Macherey-Nagel (Düren, Germany) fused-silica (30 m × 0.25 mm i.d.) column coated with 14% cyanopropylphenyl, 86% dimethylpolysiloxane (1701, film thickness 0.25 µm) and with a carrier gas helium at 2.0 mL/min constant flow. During the adsorption phase the oven was kept at 38 °C, then increased up to 245 °C at a rate of 10 °C/min.

The mass spectrometry interface was kept at 260 °C, the ion source operated at 180 °C in electron impact mode at 70 eV and MS analyzer in full scan mode from 41 to 400 m/z.

The compounds’ identification was performed by mass spectra interpretation, by comparison with the NIST20 computer library and the literature data [47]. Semiquantitative analysis (% relative concentration) was performed using the same Konik instrumentation and the same GC and TOTAD conditions. The mass spectrometric detector was replaced with a flame ionization detector (FID) operating at 280 °C. The analyses were carried out in triplicate.

3.3. DI-SPME/GC-MS Analysis of C. sativa Hy

To describe the chemical profile of the headspace from the Hy, direct immersion solid-phase microextraction (DI-SMPE) sampling followed by GC-MS technique was performed. About 2.0 mL of Hy was placed into a 7 mL glass vial with a PTFE-coated silicone septum. In order to reach thermal equilibrium, a thermostatic bath for 15 min with constant magnetic stirring was utilized. For the extraction of compounds, a SPME device from Supelco (Bellefonte, PA, USA) with 1 cm fiber coated with 50/30 μm DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) was chosen [48]. Before use, the fiber was conditioned at 270 °C for 30 min. After conditioning, it was immersed directly in the aqueous solution for 20 min at 60 °C for the extraction of volatile and semi-volatile compounds. For the extraction of the adsorbed compounds, the fiber was then inserted into the injector of the GC maintained at 250 °C. A Clarus 500 model Perkin Elmer (Waltham, MA, USA) gas chromatograph coupled with a mass spectrometer and equipped with an FID (flame detector ionization) was used for the analyses. For the separation of compounds, a Varian Factor Four VF-1 (60 m × 0.32 mm ID, DF = 1.0 μm) capillary column was housed in the GC oven. The chromatographic conditions were as follows: 50 °C up to 220 °C for 10 min at a rate of 6 °C min⁻¹. A gas carrier was used with the flow rate of 1.0 mL min⁻¹. The mass spectra were obtained in the electron impact mode (EI) at 70 eV in scan mode ranging 35–400 m/z.

The volatile compounds were identified by the comparison of the MS-fragmentation pattern of the analytes with those of pure components stored in the Wiley 2.2 mass spectra library database. Further, the linear retention indices (LRIs) were calculated using a series of alkane standards and compared with those reported in the literature. The relative amounts of the components were expressed as percent peak area relative to total peak area without the use of an internal standard and any factor correction. The analyses were carried out in triplicate.

3.4. GC-MS Analysis of C. sativa EO

The chemical characterization of C. sativa EO was obtained by GC/MS analysis using the same apparatus described above. In this case, the temperature program was set as follows: 60 °C up to 220 °C for 20 min at a rate of 6 °C min⁻¹. The identification and quantification of the components were performed as reported in the previous Section 2.3. The analyses were conducted in triplicate.

3.5. Cell Cultures

Human melanoma (A375, LOX IMVI), non-small cell lung (H1299, A549), colon (HT29) and pancreatic (L3.6) cancer cell lines were cultured in RPMI-1640 medium (Euroclone, Milan, Italy) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/mL penicillin/streptomycin (Euroclone) and 1% L-glutamine (Euroclone). Human telomerase reverse transcriptase immortalized fibroblasts (BJ-hTERT) were cultured in Dulbecco’s Modified Eagle’s medium supplemented with 10% inactivated fetal bovine serum, 100 μg/mL penicillin/streptomycin and 1% L-glutamine. Cells had been recently tested for mycoplasma contamination and authenticated through the analysis of STR profile.

3.6. Treatments and Analysis of Cell Viability

3 × 10³ cell were plated in 96 well plates and, after 24 h, were treated with increasing concentrations of EO or Hy. The latter were serially diluted in complete medium. Cell viability was evaluated by measuring 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide inner salt (MTT, Sigma-Aldrich, St. Louis, MO, USA) dye absorbance after 72 h of treatment. Experiments were repeated at least three times with six technical replicates ran on the same plate.

The authors are thankful to Aeroponica Perrotta Farm—Nola (NA) 80035, Italy for providing Cannabis sativa L. cv. Carmagnola essential oil and hydrolate.