Different relative areas were achieved for cannabidiol, cannabidiolic acid (CBDA), tetrahydrocannabinol, and tetrahydrocannabinolic acid (THCA) in every Cannabis plant extract, as indicated in the following table (Table 1).

Characterization of the cells’ viability was performed in order to determine the effect of the different treatments: the antioxidant compound α-tocopherol (1 μM), synthetic cannabinoids AM281 (3.5 μg/mL), and WIN55,212-2 (3.5 μg/mL), or a combination of these.

The cells treated with WIN5,212-2 reached a cell death percentage of 76.0% ± 2.0, whereas AM281, an antagonist of the CB1 receptor, achieved 15.5% ± 1.6 cell death. When both of the cannabinoid molecules were incubated together, a partial reduction in cell death (58.5% ± 5.36) was observed, compared to that produced by WIN55,212-2 alone (Figure 1).

The α-tocopherol pre-treatment reduced the cell death triggered by WIN55,212-2—it dropped from 76.0% ± 2.0 to 37.0% ± 4.2. Moreover, this reduction in the WIN55,212-2-evoked cell death was higher when α-tocopherol and AM281 were used together, at 18.0% ± 1.0. This was similar to the cell death obtained when AM281 or α-tocopherol were assayed individually (15.5% ± 1.5 and 19.5% ± 2.5, respectively).

Regarding the effects of the Cannabis extracts from different plant varieties, the cell death of the neuroblastoma cell cultures was monitored every 24 h during their treatment with the plant extracts. This was both with the presence and absence of 3.5 μg/mL AM281 and 3.5 μg/mL WIN55,212-2, and both with and without pre-treatment with 1 μM α-tocopherol.

The exposure of neuroblastoma cells to a D2-7 extract, at a concentration of 3.5 μg/mL, reached 72% ± 9 cell death (Figure 2A), whereas the same concentration of the extracts of P0-3 and D2-2 reached 50% and 28%, respectively. The differences between WIN55,212-2 and the extracts were only reached in the case of D2-2, which achieved 40% less cell death. Moreover, a positive correlation was also observed between the tetrahydrocannabinol content of an extract and neuroblastoma cell death (Figure 2B).

Significant differences were observed between the D2-7 extracts and the others in the presence of AM281. Under this condition, the D2-7 extracts achieved a cell death rate of 60% ± 1, which was 12% lower than that achieved by the control group. P0-3 showed a different effect to D2-7 but not D2-2. The P0-3 cannabinoid treatment achieved 18% ± 3.8 cell death, which was almost half the level of neuroblastoma cell death that was observed in the control (32% less cell death). Moreover, D2-2 only reached 12% ± 2 cell death, which was 16% less than the control.

The α-tocopherol pre-treatment reduced the cell death rate in all cases, and no differences among the plant extract treatments were observed, with cell death rates of 17% ± 1, 12% ± 1, and 21% ± 7 in D2-7, P0-3, and D2-2, respectively. A reduction in cell death was observed in every plant extract, with a decrease of 76.3% in D2-7, 76% in P0-3, and 25% in D2-2. Moreover, a combination of both α-tocopherol pre-treatment and AM281 treatment, alongside the different extracts, presented differences between D2-7 and P0-3. The two cannabinoids reached 46.5% ± 5.5 and 16.5% ± 0.5, respectively, while D2-2 did not show any significant differences, reaching 29% ± 8 cell death (Figure 3). In addition, the α-tocopherol pre-treatment used alongside the AM281 treatment decreased by 17.5% in the case of D2-7 and by 17% in the case of D2-2, which was a slight increase of 1.5%.

The characterization of the plant extracts’ effects on cytochrome c oxidase activity was performed both in the absence and the presence of plant extracts at different concentrations (1 μg/mL, 10 μg/mL, and 100 μg/mL). This was performed in order to elucidate whether or not the differences in the cell mortality rates could be due to their effects on cytochrome c oxidase (Figure 4A). The enzymatic activity was measured for 16 h, using a cell homogenate in a reaction solution and without plant extracts as a control. Cytochrome c inhibitions of 33.07% ± 2.03, 23.96% ± 5.09, and 19.35% ± 3.12 were achieved with D2-7, P0-3, and D2-2, respectively, at concentrations of 100 μg/mL (Figure 4B). Moreover, there was found to be a positive correlation between the quantity of tetrahydrocannabinol and cell death. The quantity of cannabidiol did not show a correlation with cell death.

The effects of the plant extracts on the cell homogenates’ lipidome were studied by analyzing the lipid fingerprints of the cell homogenates that were exposed to the Cannabis sativa plant extracts, both with and without α-tocopherol pre-treatment. A distinguishable lipid fingerprint was observed because of their exposure to the extracts. In addition, differences were observed among the extracts (Figure 5).

Regarding the glycerophospholipids, significant differences were observed among several long-chain PCs—between 32 and 40 carbons—in the cells that were exposed to the extracts, compared to controls. PCs with more than 36 carbons generally increased: the higher the number of carbons, the higher the relative enhancement. However, this effect seemed to be reduced when the unsaturation number increased, e.g., PC 40:4 reached 423% when exposed to D2-7, whereas PC 40:5 and PC 40:6 only reached 173% and 123%, respectively. A similar effect was observed in the case of PC 38:4 and PC 38:6. By contrast, PCs with less than 36 carbons generally decreased, except in the case of PC 32:0.

In the case of the PA species, a clear difference was observed between D2-7 and the other extracts. While the D2-7 extract generally triggered a decrease, P0-3 and D2-2 induced an upregulation. This discrepancy was particularly high in PA 32:0 (Figure 5A). A similar effect was observed in the PE species and their ether versions, in which the D2-7 extract generally promoted a reduction in these lipid species, whereas, with the other plant extracts, they increased, except in the case of PE 34:2 and PE 38:4. Moreover, this discrepancy between the D2-7 extract and the others was also found in the following phosphoinositols: PI 36:2, PI 36:4, PI 38:4, and PI 40:4. The only exception here was for PI 38:5, which was reduced by all three of the extracts assayed. In contrast, lysophosphatidilinositol 15:0 was enhanced in the cells exposed to D2-7, whereas, with the other extracts, it was unchanged or even slightly decreased. This Lyso-PIP2 contains pentadecanoic fatty acid.

Regarding sphingolipids and ceramide phosphates, the three extracts appeared to promote a reduction in SM d34:1, SM d36:1, SM d42:2, CerP d36:2, and CerP d38:2. The D2-7 extract was the one that induced the greatest effect (Figure 5B).

The α-tocopherol pre-treatment in the cell lines exposed to these plant extracts led to lower cell mortality rates at 24 h. Thus, this decrease in cell mortality might entail changes in the lipidic fingerprints of the cell homogenates. Different effects on the lipid relative abundance were observed for every Cannabis plant extract (Figure 6).

Significant changes were detected in the glycerophosphocholine species with between 30 and 38 carbons, with respect to the groups that were exposed to the antioxidant pre-treatment compared to the groups that were only treated with the extracts. This change in relative abundance was different depending on the extract used: there was a decrease in the cells exposed to the P0-3 extract, whereas an increase was observed in the D2-7 and D2-2 extracts. However, an exception was seen in PC 34:1 and its ether version, which were reversed. In regard to the PA species, the α-tocopherol pre-treatment reduced PA 36:1 and PA 32:1 in the cultures treated with the D2-2 and P0-3 extracts. In particular, PA 32:1 was absent in the pre-treated samples that were exposed to P0-3 (black arrow in Figure 6A). The opposite effect was observed in the cells that were exposed to D2-7, in which an increase in PA 32:1 and PA 36:1 was detected in the α-tocopherol pre-treated samples. Furthermore, PA 32:1 was only present in the cells that were pre-treated with α-tocopherol and exposed to D2-7, and not in those that were treated with this extract alone, i.e., without the antioxidant pre-treatment (red arrow in Figure 6A).

As in the case of the PA species, a similar effect was observed in the phosphoethanolamine species and their ethers, phosphoglycerol, and phosphoinositols with α-tocopherol. These lipid species were only present in the cells that were exposed to the D2-7 extract and pre-treated with α-tocopherol, whilst they decreased in those exposed to P0-3 and D2-2 extracts. In contrast, various lysophosphatidylinositol species displayed a general increase in the pre-treated cells that were exposed to the P0-3 extract, and a general decrease in the pre-treated cells that were exposed to D2-7. No general trend was observed in the cells that were treated with α-tocopherol and the D2-2 extract. In particular, when using this extract, a decrease was found in Lyso-PIP 13:0, an increase was found in Lyso-PIP O-14:0, and no change was found in Lyso-PIP2 15:0, compared to the group without antioxidant pre-treatment.

(±)-α-tocopherol; methyl viologen dichloride hydrate; DMEM:F12 medium; penicillin–streptomycin solution hybri-max (P/S); fetal bovine serum (FBS); L-glutamine (L-Glut); non-essential amino acid solution (NEAA); trypan blue; AM281; WIN55,212-2; mesylate salt; 2-mercaptobenzothiazole; and 1,5-diaminophtalene were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Methanol and acetonitrile were purchased from Panreac (Barcelona, Spain) and cesium chloride was purchased from HoneyWell (Charlotte, NC, USA).

The extracts were reconstituted with absolute methanol to a concentration of 1 mg/mL and analyzed by HPLC–PDA in a Shimadzu unit with an LC-20AD pump and a CTO-10AS VP column oven, coupled to an SPD-M20A Diode Array Detector. Separation was obtained using an ACE 3 C18 column (150 mm × 4.6 mm, 3 µm particle size) with an ACE3 C18 analytical pre-column. Compounds were eluted with methanol 0.1% acetic acid (LC–MS grade) (MeOH): MiliQ water 0.1% acetic acid, with a gradient of 80:100% over 15 min, 100% MeOH over 5 min, and 100:80% for 10 min, with a flow of 0.5 mL/min. The results were analyzed at a UV wavelength of 210 nm. The stock solutions of the ethanolic extracts were injected at 1 mg/mL, carrying out a 10 µL injection through an automatic injector (SIL-20A XR, Shimadzu, Kyoto, Japan). All extracts were dissolved in 100% MeOH for injection. The identification of the products was carried out through a comparison between the retention time and UV spectrum of standards of the pure product, previously injected. The percentage expressed in the table of the products is the relative percentage of each product of the whole extract, excluding the peaks of the sample with a percentage lower than 0.011%.

The human neuron cell line, SHSY-5Y, was cultivated in 12-well plates in complete medium (DMEM:F12 medium; 15% fetal bovine serum (FBS); 1% L-glutamine (L-Glut); 1% of non-essential amino acid solution (NEAA); and 1% penicillin–streptomycin solution hybri-max (P/S)) for 24 h, at 37 °C, 5% CO₂, and constant humidity. Cells were habituated to low-serum conditions (DMEM:F12; 0.2% charcoal-treated FBS; 1% L-Glut; 1% NEAA; and 1% P/S) for 12 h before treatments began. After adaptation, different concentrations of cannabinoid extract were tested (1 μg/mL; 10 μg/mL; 30 μg/mL; and 100 μg/mL). In addition, a pre-treatment of 3 h, with or without α-tocopherol (1 μM) prepared in low-serum medium, was performed before treatment with or without cannabinoid extracts (3.5 μg/mL of plant extract concentration) in low-serum conditions, for 24 h. Moreover, cells were treated with synthetic cannabinoids WIN55,212-2 (3.5 μg/mL) or AM281 (3.5 μg/mL), with or without the α-tocopherol pre-treatment (1 μM). Cannabinoid extracts were diluted in absolute ethanol, while synthetic cannabinoids and α-tocopherol were diluted in DMSO. All tested situations had the same concentration of DMSO and absolute ethanol, to avoid changes due to vehicle solutions. After 24 h of cannabinoid treatment (either synthetic or plant extract), the viability assays were performed. Samples with only α-tocopherol pre-treatment were incubated with fresh low-serum medium, in the same manner as treated ones.

To analyze the cellular viability at every condition and time point, trypan blue viability assays were performed. Cells were detached from the culture well using a mechanical method. The cell suspension was diluted 1:1 with 0.4% trypan blue solution, and cells were counted using a Neubauer chamber in an inverted microscope: Olympus CKX41 (Olympus Corporation, Tokyo, Japan). Dead cells (with a compromised membrane) were stained dark blue. The percentage of dead cells with respect to total cells was calculated.

Viability data handling and analysis was carried out using Excel and GraphPad software (version 9.2). Briefly, cell viability data were presented as a percentage of cell growth. The identification of outliers was carried out by applying the following formulas:

SD—standard deviation; DF—deviation factor.

Points were identified as outliers and excluded if Y₁ was higher than the point analyzed, or Y₂ was lower than the point examined. We used a deviation factor of 1.25 in our analysis. Data were expressed as means of independent data points ± S.E.M. The results were analyzed using one-way, two-tailed, two-way ANOVA, with Tukey’s post-hoc. Statistical differences were indicated with p-values ≤ 0.05.

The human neuron cell line, SHSY-5Y, was cultivated in 75 cm² well plates, in complete medium (DMEM:F12 medium; 15% fetal bovine serum (FBS); 1% L-glutamine (L-Glut); 1% of non-essential amino acid solution (NEAA); and 1% penicillin–streptomycin solution hybri-max (P/S)), until they reached confluency. Cells were detached by a mechanical method to avoid enzymatic treatments (medium was changed to PBS at 4 °C and flasks were tapped laterally). Once cells were detached, they were pelleted and stored at −80 °C until use. The cell pellet was resuspended in phosphate buffer, pH 7.4, and passed through a 30Gx1/2″ syringe (0.3 mm × 12 mm), and then sonicated using a bath sonicator three times, for 10 s, to obtain the cell homogenate, and adjusted to 0.2 mg/mL solution in phosphate buffer (0.2 M, pH 7.4). The cell homogenate was added to the reaction solution (DAB 0.5 mg/mL and cytochrome C 0.01%) in a 96-well plate, in the presence or absence of Cannabis sativa plant extracts (final concentrations of the whole extract: 1 μg/mL, 10 μg/mL, and 100 μg/mL; concentrations of CBD and THC are indicated in Table 2). The reaction was measured at 450 nm, every 5 min, for 16 h. The linear part of the reaction curves was analyzed and slopes were obtained for each concentration.

Cells were detached by a mechanical method to avoid enzymatical treatments (medium was changed to PBS at 4 °C and flasks were tapped laterally). The cell suspension was passed through a 30Gx1/2″ syringe (0.3 mm × 12 mm) to produce a cell homogenate and then pelleted. A matrix-saturated solution was prepared by diluting 2-mercaptobenzothiazole (MBT) in a solution 3:5 of CsCl aqueous 200 mM:MeOH (final concentration of CsCl 75 mM). For positive-ion mode and 1,5-diaminopthtalene (DAN), a 1:1 solution of acetonitrile: distilled water was used. For each ionization mode, 2 μL of cell homogenate was added to 20 μL of matrix solution, 1 μL of each of this solution was spotted onto a sample plate, and each sample was spotted in triplicate.

The mass spectrometer used was an LTQ-Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a MALDI source with a N₂ laser (60 Hz, 100 μJ/pulse maximum power output). The laser spot was an ellipsoid of approximately 50–60 μm × 140–160 μm. Two microscans of 10 shots/spot were used, with a laser power output of 20 μJ for MS+ and 30 μJ for MS𲈒. Data loading included spectra normalization by total ion current (TIC), spectra alignment, and peak picking, filtering all the m/z with intensity <0.5% of the strongest peak in the spectrum.

MALDI spectra were aligned using MATLAB (Matworks, Natick, MA, USA), and lipids’ assignment was performed using a homemade database and the Lipid Maps LMSD database. For MALDI data analysis, MS+ and MS− data were normalized separately and then analyzed together. Matrix peaks and isotopic distribution were removed, and the remaining peaks were normalized against their total ion current (TIC). MS+ and MS− data were standardized using the z-score method, using the following formula.

x—observed value; μ—mean of the sample; σ—standard deviation of the sample.

The data supporting the findings of this study are available from the corresponding author, Gabriel Barreda-Gómez, upon reasonable request.