Mosquitocidal and Anti-Inflammatory Properties of The Essential Oils Obtained from Monoecious, Male, and Female Inflorescences of Hemp (Cannabis sativa L.) and Their Encapsulation in Nanoemulsions

2.2. Toxicity of C. sativa Essential Oils on Mosquito Larvae and Pupae

An. stephensi and An. gambaie larvae and pupae were exposed to different dilutions of the three hemp EOs obtained from the monoecious Felina 32 and female and male inflorescences of CS, and the mortality was recorded after 24 h of treatment. The dose–response curves showed an insecticidal effect of the three EOs on larvae and pupae of An. stephensi (Figure 2) and An. gambiae (Figure 3). No effects were revealed when the mosquitoes were exposed to pure breeding in water or DMSO (control groups).

A dose–response effect was observed in the insecticidal activity of the three EOs in both An. stephensi and An. gambiae. In An. stephensi, mortality caused by hemp EO at 100 ppm was 82.7% and 100% (Felina 32), 90.2% and 94.2% (CS female), and 89.8% and 90.5% (CS male) on larvae and pupae, respectively. At the same concentration (100 ppm), the mortality produced on An. gambiae was 91.1% and 84.9% (Felina 32), 91.6% and 79.6% (CS female), and 89.8% and 79.7% (CS male) on larvae and pupae, respectively. Mortality of 100% was observed when the mosquitoes (L3 and pupae) were exposed at the maximus dilution of the three EOs.

LC₅₀ values were in the range 73.50 to 78.80 ppm for larvae and 20.13 to 67.19 ppm for pupae, in An. stephensi and An. gambiae, respectively. Results for each EO showed a lower susceptibility of larvae compared to pupae (Table 2).

2.3. Effect of Hemp Essential Oils in HaCaT and NHF A12 Cell Lines

The cytotoxic effect of hemp EOs (derived from male and female inflorescences of cv CS) was evaluated in HaCaT (human keratinocytes) and NHF A12 fibroblasts cell lines by MTT assay (Figure 4). Cells were treated with different dilutions, from pure (1040 mg mL⁻¹) to 0.163 mg mL⁻¹, for 24 h. EO from male CS inflorescence showed higher cytotoxicity than EO from female CS. However, NHF A12 were more resistant (Figure 4). The IC₅₀ values for the EO from male CS inflorescence were 2.23 ± 0.09 and 3.71 ± 0.1 mg mL⁻¹, on HaCaT and NHF A12 cells, respectively, while for the EO from female CS inflorescence, the IC₅₀ values were higher than 5.2 mg mL⁻¹. To further evaluate the potential anti-inflammatory effects of hemp EOs, we firstly tested the ones obtained from male and female inflorescences of cv CS in both cell lines, at 0.65 mg mL⁻¹.

2.4. Preparation and Characterization of C. sativa Essential Oil Nanoemulsions

NEs are colloidal systems that offer the great advantage of being able to encapsulate a higher amount of oil phase compared to similar nanosystems, i.e., microemulsions. Moreover, they require a very low amount of surfactant with a SOR (surfactant–oil ratio) recorded between 1 and 2, compared to that of microemulsions, which is generally higher than 2 (SOR > 2). However, NEs are energetically disadvantaged nanosystems; thus, an external energetic input is required for their formation. In this respect, for the achievement of EO NEs, samples were subjected to a pressure of 130 MPa, selected after the evaluation of different operating pressure, from 50 to 150 MPa.

After a preliminary screening using a model oil phase, the quantitative composition of the NEs has been selected as follows: 6% (w/w) of the oil phase was emulsified in the aqueous medium through the addition of 4% (w/w) of surfactant, Tween 80. However, this composition was not suitable for the encapsulation of the EO from female inflorescences of cv CS; in fact, formulations A8 and A9 recorded a high PDI (polydispersity index) value at t0, indicating the polydispersity of the size distribution, with a particle population with a mean diameter higher than 500 nm. Moreover, after 1 month of storage, instability phenomena, i.e., creaming and phase separation, occurred in such samples (Table 3).

In this respect, the EO percentage was decreased to 4% (w/w) in order to obtain a stable monodispersed system, having a mean diameter within 200 nm and a PDI value lower than 0.3. Although the reduction of the oil phase led to slight system improvement, samples A8B and A9B revealed their instability between 1 and 2 months after their preparation, undergoing phase separation (Table 3).

Then, we decided to add ethyl oleate, commonly used as an oily phase in such systems, to overcome instability issues related to the physicochemical properties of the EO. In fact, ethyl oleate showed good solvent properties, making the oil phase more homogeneous by allowing a better dispersion and encapsulation of active ingredients. The minimum EO–ethyl oleate ratio allowing a good dispersion of the EO in the oil phase was fixed at 1:1. Thus, keeping the amount of oil phase constant at 6% (w/w), systems were composed of 3% EO, 3% ethyl oleate, 4% Tween 80, and water (Table 3).

Samples A10 and A21 showed a monomodal size distribution with a size in the nanometric range, below 200 nm, which is the limit established by some authors for the definition of a nanosystem.

4.2. Reagents

Etoposide (ETO) was purchased from Abcam (Milan, Italy) and diluted in sterilized water at a concentration of 50 mM. Hemp EOs used are those from monoecious inflorescence of Felina 32 and from male and female inflorescences of CS. Each EO was used pure or diluted in DMSO for the analysis.

4.3. Mosquitoes

An. gambiae (G3 strain) and An. stephensi (Liston strain) were reared in the insectary at 29 °C ± 2 and 85% ± 5 relative humidity, with photoperiod (12:12 light:dark). Adult insects were maintained with 5% sucrose solution ad libitum, and adult females were fed on mouse blood for egg-laying. Eggs were collected on wet filter paper for 48 h before hatching. Larvae were maintained in spring water and fed daily with commercial fish food.

4.4. Cell Lines

An immortalized human keratinocyte (HaCaT) cell line (Creative Bioarray, Shirley, NY, USA) was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU mL⁻¹ of penicillin, 100 µg of streptomycin (Lonza, Allendale, NJ, USA), 1 mM sodium pyruvate (Lonza) and maintained at 37 °C with 5% CO₂ and 95% humidity. Primary human stabilized NHF A12 fibroblast cell line (ATCC, LGC Standards, Milan, Italy) was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU mL⁻¹ of penicillin, 100 µg of streptomycin (Lonza), 1 mM sodium pyruvate (Lonza) and maintained at 37 °C with 5% CO₂ and 95% humidity.

4.5. Steam Distillation

After being cut into small parts, 2500 g of fresh male inflorescences of cv CS were subjected to steam distillation (SD), by an Albrigi Luigi E0106 (Stallavena di Grezzana-Verona, Italy) stainless steel apparatus (capacity 20 L) for 4 h, with 2 L of distilled water at the bottom of it, to produce the steam. This device was equipped with a steel Clevenger-type apparatus and a glass burette for the collection of the EO. Once separated from the aqueous layer, the EO was collected in dark vials sealed with PTFE-silicon septa, and stored at 4 °C until analysis. The EO yield was calculated on a dry weight basis.

4.6. Hydrodistillation

Hydrodistillation (HD) was performed for dried samples of cv Felina 32 monoecious inflorescences and cv CS female inflorescences. In both cases, we used 2000 g of plant material soaked in a 20 L glass flask filled with 11 L of water that was heated by a mantle system Falc MA (Falc Instruments, Treviglio, Italy) for 4 h. The EOs were recovered by a glass Clevenger-type apparatus. In this case, the obtained EOs were kept in the dark glass vials in the fridge before chemical characterization and biological assays. The EO yield was calculated on a dry weight basis.

4.7. GC-MS Analysis

The chemical composition of hemp EOs was analyzed through an Agilent 6890N GC-MS system coupled to a 5973N single quadrupole detector mass spectrometer. Separation was provided by a HP-5MS capillary column (5% phenylmethylpolysiloxane, 30 mL × 0.25 mm i.d., 0.1 µm f.t., Agilent, Santa Clara, CA, USA). The temperature program was as follows: 60 °C for 5 min, then 4 °C min⁻¹ up to 220 °C, finally 11 °C min⁻¹ to 280 °C, maintained for 15 min, for a total run time of 65 min. The temperature of the injector and detector was 280 °C. Helium (He) was the carrier gas, with a flow rate of 1 mL min⁻¹ and a 1:50 split ratio. The chromatograms were acquired in full scan in the range 29.0–400.0 uma, using electron-impact (EI, 70 eV) mode. Dilution 1:100 of essential oils in n-hexane was injected (2 µL) into the GC-MS system. The MSD ChemStation (Agilent, Version G1701DA D.01.00) and the NIST Mass Spectral Search Program were employed for the data analysis. The identification of the principal compounds was achieved by the correspondence of retention indices and mass spectra to those of ADAMS, NIST 17, FFNSC2, and WILEY 275 libraries. Furthermore, the analytical standards available in the laboratory (Sigma-Aldrich, Milan, Italy) were used for further confirmation. The relative peak area percentages were obtained by area normalization without using correction factors [49].

4.8. Insecticidal Assays

The larvicidal effect of the EOs was evaluated according to standard procedures established by the WHO [50]. Three different hemp EOs were analyzed: (i) Felina 32 dioecious inflorescence, (ii) CS female inflorescence, (iii) CS male inflorescence. For the treatment of mosquitoes, the EOs were dissolved in dimethyl sulfoxide (DMSO) 1:10. Twenty-five larvae L3 (third instar stage) and twenty-five pupae were placed in plastic cups containing water. Treatment was carried out using different dilutions of the three essential oils preparations: 50, 75, 100, and 125 ppm. Control groups were prepared adding pure breeding water or DMSO (200 µL). Mortality of larvae and pupae was observed after 24 h. Three independent experiments were performed.

4.9. MTT Assay

Three thousand cells per well were seeded in 96-well plates. After one day of incubation, hemp EOs (from male and female inflorescence of CS) and vehicles were added, and six replicates were used for each treatment. At the indicated time point, 24 h, cell viability was investigated by adding 0.8 mg mL⁻¹ of 3-(4,5-dimethylthiazol-2yl)-2,5 diphenil tethrazolium bromide MTT (Sigma Aldrich) to the media. The reaction was given by mitochondrial reductase which changed MTT (tetrazolium salt) colour from yellow to purple, forming formazan crystal. After 3 h, plates were centrifuged, the supernatant was removed, and the pellet of salt crystals was solubilised with 100 µL well⁻¹ of DMSO. The absorbance of the sample against a background control (medium alone) was measured at 570 nm using an ELISA reader microliter plate (BioTek Instruments, Winooski, VT, USA).

4.10. Gene Expression Analysis by TaqMan Array

Total RNA was extracted with the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reactions (qRT-PCR) were performed with TaqMan Array (Thermo Fisher) using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR parameters were 10 min at 95 °C followed by 40 cycles at 95 °C for 15 s and 60 °C for 40 s. The relative amount of target mRNA was calculated by the 2^−ΔΔCt method, using GAPDH as a housekeeping gene. All samples were assayed in triplicates on the same plate. Measurement of GAPDH levels was used to normalize mRNA contents, and target gene levels were calculated by the 2^−ΔΔCt method. The gene list is presented in Figure 6.

4.11. ELISA Assay

The concentration of inflammatory cytokines released from HaCaT and NHF A12 cell lines after treatment with ETO combined and not with hemp EOs, was evaluated in cell culture supernatants of cells treated for 24 h. The levels of cytokines were evaluated in duplicated and in two independent experiments using ELISA kit (Abcam). Cytokine concentrations were calculated by plotting the OD values in the equation curve obtained with standard, according to the manufacturer’s protocol.

4.12. Alkaline Comet Assay

Cells were plated in six-well plates (3 × 10⁴ cells well⁻¹) one day before treatment exposure. Semiconfluent cultures were treated for 24 h with hemp EOs from CS male and female inflorescences (0.65 mg mL⁻¹) and 10 µM of ETO. Cells treated with vehicle were included in all series. The Comet assay was performed under alkaline conditions following the ABCAM protocol. Briefly, after exposure to treatments, the cells were resuspended in 1× PBS and added to 75 µL of molten (37 °C) 0.5% low-melting-point agarose gel to achieve a cell concentration of 1 × 10⁵ cells mL⁻¹. The agarose was pipetted onto the Comet slides. Slides were stored in the dark at 4 °C for 10 min before adding prechilled lysis buffer for 45 min at 4 °C in the dark. The slides were immersed in freshly prepared alkaline solution (0.25 M NaOH containing 0.1 µM EDTA, pH 12.6) for 30 min at the same conditions. Slides were then removed and washed twice with TBE buffer for 5 min. Gel electrophoresis was performed at 1 V cm⁻¹ for 20 min (running amperage 3–5 mA with the distance between the two electrodes of 25 cm). The Comet slides were washed with 70% ethanol for 5 min and air-dried for 1 h at room temperature. A total of 100 µL of diluted SYBR Green solution was placed onto each dried agarose circle. The slides were then read with a fluorescence microscope (LEIKA).

4.13. Statistical Analysis

LC₅₀ values were obtained by nonlinear regression using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The graphs related to the mortality effect of the EOs on larvae and pupae of An. stephensi and An. gambiae. Data were plotted using STATISTICA software version 6.0. The correlation between the mortality and the EOs doses was assessed using linear regression analysis (Fisher–Snedecor test) using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). For MTT, RT-PCR, and Elisa assay, the data presented represent the mean and standard deviation of at least two independent experiments. The statistical significance was determined by Student’s t-test and by one-way ANOVA; *, # p < 0.01 or p < 0.05 (as described in the figure’s legends). The statistical analysis was performed using Prism 5.0a (Graph Pad).

4.14. Preparation of EO-Based Nanoemulsions

NEs were obtained through a high-energy method, by using a high-pressure homogenizer. Briefly, different percentages (6%, 4% w/w) of CS female inflorescence EO or a 6% (w/w) of a binary mixture of EO and ethyl oleate (1:1 ratio) were added dropwise to a 4% (w/w⁻¹) of surfactant (Tween 80) aqueous solution under high-speed stirring (Ultraturrax T25 basic, IKA^® Werke GmbH & Co.KG, Staufen, Germany) for 5 min at 9500 rpm. The obtained emulsions were then subjected to the homogenization process by means of French Pressure Cell Press (American Instrument Company, AMINCO, MY, USA) for four cycles at the pressure of 130 MPa.

Authors wish to thank the farm La Biologica for furnishing the plant material.