Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

2.2. CBDA Cytotoxicity in HaCaT Cells

In order to hypothesize the nutraceutical use of CBDA for skin, the cytotoxic effect of CBDA was preliminarily evaluated by means of three different assays towards the HaCaT cell line, which derives from normal human skin and it is spontaneously immortalized [28]. Evaluating CBDA cytotoxicity in HaCaT cells at ten different concentrations (from 0.1 to 100 μM), it was observed that it was able to exert a dose-dependent inhibition of mitochondrial redox activity. The compound appeared to be non-toxic when tested at low concentrations (up to 10 μM), whereas it was able to reduce dehydrogenase activity by 40.7%, 57%, and 84% at 25, 50, and 100 μM (Figure 2A). This was not in line with Martinenghi et al. [29], who observed that CBD showed dose-dependent killing up until 16 µg/mL, where at higher doses the cytotoxicity was stabilized at 50%. The increase in concentration appeared to affect cell morphology, pointing that in dosages above 10 μM, cells were similar to balloon-like bubbles [30].

When sulforhodamine B assay was performed, CBDA at 10 and 25 μM was observed to decrease by 26.5% and 35.7%, respectively, the total protein mass, which is related to cell number, whereas at the lowest tested dose there was no inhibition of cell viability (Figure 2B). However, the inhibitory effect does not increase in proportion to the dose increase, with an inhibition of 55.3% occurring in cells treated with CBDA at 100 μM. At this latter concentration, as well as at 50 μM, a marked lactate dehydrogenase (LDH) release was further detected (Figure 2C). The intracellular LDH enzyme was from damaged cells and its content is an indicator of cell membrane integrity. The effect observed in the highest concentration of CBDA-tested HaCaT cells was unrelated to what was recently found testing an extract of Cannabis sativa L. herb [31]. The neutral derivative of CBDA was previously tested on keratinocytes and, according to our data, no cytotoxic effects were at 1, 5, 10, and 20 μM after 6, 12, and 24 h [32]. Furthermore, it was reported that CBD was able to induce keratinocyte differentiation, and skin development [33]. The extensive investigation of the CBD efficacy in maintaining skin health [34] does not find a counterpart in its precannabinoid, which has attracted less attention, probably due to its tendency to decarboxylate or, more simply, to the lack of awareness of its presence and relative abundance. Thus, only indirect data are available on CBDA activity in skin cells. Recently, an hemp seed apolar extract, with CBD and CBDA as the main compounds, was reported to exert anti-inflammatory and anti-lipogenic activity in a Propionibacterium acnes-triggered inflammation and lipogenesis model [35]. Considering that both CBDA and CBD have structurally low binding affinity to the endocannabinoid receptors, the less polarity of CBDA was not limiting for microemulsion formulations for transdermal delivery [36].

2.3. CBDA Modulated Cytokines and Chemokines in HaCaT Cells

Since the HaCaT cell line is a reliable model to screen for new anti-inflammatory compounds for skin diseases, and keratinocytes are involved in the initiation and perpetuation of the cutaneous inflammatory response [37], the study of the ability of CBDA to differentially modulate the release of proinflammatory mediators was carried out (Figure 3A). For this purpose, HaCat cells were first treated with the non-toxic doses 2.5, 5, and 10 μM. Cytokines, including chemokines, play a critical role in mediating inflammation, and they are involved in cell communication, also regulating immunity, and cell activation, migration, and proliferation [38]. The CBDA concentration appeared to be determinant in the response observed. In fact, it was observed that CBDA inhibited dose-dependently IL-1ra (IL-1 receptor antagonist), a regulator of IL-1 signaling, and a competitive antagonist of the IL-1 agonist receptor (IL-1R1) [39]. The inhibition of the pleiotropic cytokine interleukin-9 (IL-9) also occurred. T cells and mast cell growth factor are implicated in allergic and autoimmune disorders, parasitic infections, and antitumor immunity [40]. Recently, it was observed that an increased IL-9 secretion is related to atopic dermatitis anthogenesis [41]. Moreover, CBDA was able to decrease IFN-γ, CXCL8, CXCL10, CCL2, CCL4 and CCL5. With the only exception of the effect on CXCL8, towards which a similar response was from CBDA 2.5 and 5 μM treatments, all the other mediators were extensively inhibited in a dose-dependent manner, with CBDA at 10 μM exerting the highest activity as also evidenced by the heatmap (Figure 3B). CBDA appeared to effectively exert the downregulation of IFN-γ, which is the dominant cytokine in inflamed skin, and in the skin of patients with atopic dermatitis, where it mediates also the upregulation of CCL5 expression [42]. The latter is a proinflammatory chemokine, formerly also known as RANTES, whose expression increases in lesioned skin [43], especially in lesions of psoriasis [44]. CCL5 expression detected in the supernatant of HaCaT cells was reduced by 20%, following CBDA 10 μM treatment. The role of IL-8 in psoriatic skin, as well as in atopic dermatitis, also was elucidated [45], and effect of CBDA at 10 μM, which was able to reduce IL-8 by 32%, is non-negligible. IL-8 is involved and produced in many inflammatory reactions, and keratinocytes are a rich source of it [46]. While CCL4, also known as macrophage inflammatory protein-1β (MIP-1β), was decreased by CBDA treatment in the HaCaT cells, CCL2 (MCP-1, Monocyte chemoattractant peptide-1), a chemokine acting as mediator in allergic rhinitis and psoriasis [47], and able to mediate inflammation after tissue damage in various organs, was completely pulled down, with a three-fold decrease compared to untreated cells already following the treatment with CBDA 2.5 μM. Finally, a marked decrease was for CXCL10, claimed as marker of vitiligo and psoriasis [48,49]. Contrary to what previously demonstrated for CBD, an increase in GM-CSF was found, which was poorly contained in untreated cells, as well as in CBDA 2.5 and 5.0 μM treated cells. Vascular endothelial growth factor (VEGF), which was reported to modulate HaCaT cells adhesion [50], appeared to be stimulated by the highest CBDA concentration, which further slowed down the platelet-derived growth factor (PDGF), commonly involved in epithelial hyperproliferation [51]. Recent findings showed a similar cytokine and chemokine behavior in CBD-treated blood of depressed patients, concluding that cannabidiol exerts dose-dependent immunomodulatory effects, and that at higher concentrations it was able to worsen inflammation [52].

2.4. Antibacterial Activity of CBDA: MIC and MBC Determination

Disturbance in the inflammatory and immune systems mediated by the above-considered cytokines and chemokines, leads very often to episodes of opportunistic skin bacterial infections compromising skin health [53]. There is the urgent need to discover compounds acting on both the inflammatory response and the prevention of bacteria colonization. Therefore, the antibacterial activity of CBDA, also considering recent literature data [29,54] was tested ranging from 0.064 to 64 µg/mL against two Gram-negative bacteria such as P. aeruginosa ATCC 27853 and E. coli ATCC 13762 and two Gram-positive bacteria such as S. aureus ATCC 6538 and E. faecalis ATCC 29212. The MIC values of CBDA ranged from 0.5 to 4 µg/mL for all the microorganisms tested, with the exception of P. aeruginosa that is resistant up to a concentration of 8 µg/mL (Table 1). CBDA also showed bactericidal effects against both Gram-negative and Gram-positive bacteria used for the study as no growth of bacteria was observed at the different concentrations reported in Table 1 (MBC values).

2.5. Evaluation of Antimicrobial Activity of CBDA by Time-Killing Assay

The time killing studies of CBDA against E. coli ATCC 13762, S. aureus ATCC 6538 and P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212 were performed. Different CBDA concentrations were used (i.e., MIC, 2XMIC, 4XMIC values), and bacteria were analyzed at different incubation times (1, 2, 3, 5 h); data obtained are shown in Figure 4.

During the first incubation hours (2 and 3 h), CBDA exhibited a strong reduction of the growth of E. coli ATCC 13762, S. aureus ATCC 6538, E. faecalis ATCC 29212 and P. aeruginosa ATCC 27853 strains at concentrations of CBDA as shown by the MIC value (Figure 4A–D). At prolonged incubation times, a slight recovery of growth for the following three bacterial strains was observed for the MIC value: E. coli ATCC 13762, S. aureus ATCC 6538, and P. aeruginosa ATCC 27853 (Figure 4A–C). On the contrary, for E. faecalis ATCC 29212, no growth recovery was observed (Figure 4D). Regarding concentrations of CBDA of 2X and 4X MIC, at prolonged incubation times a recovery of growth and vitality was observed only for E. coli and S. aureus (Figure 4A,B).

2.6. Biofilm Formation

CBDA was tested for inhibition of biofilm formation using two different assays, quantified through staining with crystal violet (Figure 5A) and staining and visualization with a microscope (Figure 5B). Different concentrations (MIC, 2X MIC, 4X MIC) of CBDA were used, as shown in Figure 5A,B. CBDA was able to inhibit biofilm formation from all bacteria strains at concentrations as low as the MIC value (Figure 5A). In addition, during the biofilm formation phase, CBDA showed maximum activity at concentrations corresponding to 4X MIC for each microorganism (Figure 5A). Biofilms formed by S. aureus and P. aeruginosa after 24 h of static culture were stained with a LIVE/DEAD stain and imaged by Cell Discoverer 7, Zeiss (Figure 5B). Upon examination of the images, the biofilms formed by untreated bacteria were noticeably thicker and bigger in volume than that those formed by treated bacteria.

The inhibition of biofilm formation induced by CBDA was evident at concentrations equal to the MIC and the maximum effect was obtained at concentrations corresponding to 4X MIC for both S. aureus and P. aeruginosa. The antibiofilm formation of cannabinoids was shown for CBD, which inhibited Candida albicans biofilm by a multi-target mode of action [55], and in S. aureus, S. pneumoniae, and C. difficile, acting mainly defining membrane disruption [56].

3.2. Cell Culture

Human HaCaT epidermal keratinocyte cell line was purchased from ATCC (American Type Culture Collection). Cells were cultured in DMEM high glucose medium supplemented with 10% Fetal Bovine Serum, 50.0 U/mL penicillin, and 100.0 μg/mL streptomycin, at 37 °C in a humidified atmosphere containing 5% CO₂.

3.3. MTT Cell Viability Assay

Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [12]. HaCat cells were seeded at 1.5 × 10⁴ cells per well in 96-well plates. Twenty-four hours after seeding, cells were treated with CBDA at 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 µM, in the medium for 24 and 48 h exposure times. As control, HaCaT cells were incubated in the medium. After incubation times, the cells were stained with MTT dye solution (0.5 mg/mL) and incubated for 4 h. Subsequently, the MTT-formazan crystals were solubilized in dimethyl sulfoxide (150 μL) and absorbance was read at 570 nm by using a Tecan SpectraFluor (Männedorf, Switzerland) fluorescence and absorbance reader. The cell viability was expressed as a percentage of mitochondrial redox activity (±standard deviation, SD) of CBDA-treated cells in respect to untreated control.

3.4. SRB Cell Viability Assay

Cell density determination by sulforhodamine B (SRB) was carried out seeding 1.5 × 10⁴ HaCaT cells per well in 96-well plates [57]. Twenty-four hours after seeding, cells were treated with CBDA at 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 µM (final concentration level in the volume of 100 μL), in the medium for 24 h exposure time (12 wells for each treatment). Untreated HaCaT cells were also incubated (12 wells). At 24 h of incubation, after medium removal, cells were fixed with ice-cold trichloroacetic acid (TCA, 10% (w/v), 40 µL) for 1 h at 4 °C. Then, plates were washed with distilled water, and dried. Then, 50 µL of sulforhodamine B (SRB, 0.4% (w/v) in 1% aqueous acetic acid) solution were added to each well, and the plates were incubated at room temperature for 30 min. After the unbound dye removal, by washing the plates with 1% aqueous acetic acid, plates were dried. Thus, the bound SRB was solubilized with unbuffered Tris Base (100 µL; 10 mM, pH 10.5), and the absorbance at 570 nm was measured using a Tecan SpectraFluor fluorescence and absorbance reader. The cell viability inhibition was calculated as a percentage (±standard deviation, SD) of CBDA-treated cells with respect to untreated control.

3.5. LDH Cytotoxicity Assay

Lactate dehydrogenase (LDH) activity was measured by using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Munich, Germany), according to the instructions of manufacturer. Briefly, HaCat cells were seeded at 1.5 × 10⁴ cells per well in 96 well plates. Twenty-four hours after seeding, cells were treated with CBDA at 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 µM (final concentration level in the volume of 100 μL), in the medium for 24 h exposure time (12 wells for each treatment). Untreated HaCaT cells were also incubated (12 wells). After incubation times, a high control was established by adding 10 μL of the lysis buffer to 6 wells for each treatment and to 6 wells of untreated cells. The plate was incubated at 37 °C for 30 min in the CO₂ incubator, and then the working solution (100 μL) was added. The mixtures reacted for other 30 min, after that the stop solution (50 μL) was added, and the absorbance was read at 490 nm by using a Tecan SpectraFluor fluorescence and absorbance reader. The LDH cytotoxicity was expressed using the following equation: ((test compound − low control)/(high control − low control)) × 100 [57].

3.6. Cytokine Analysis

HaCat cells were seeded at 3.0 × 10⁵ cells per well in 6-well plates. After 24 h, cells were treated with CBDA at 2.5, 5, and 10 µM concentration levels, in free-serum medium, for 48 h exposure time. Following cell harvesting, supernatants were collected and stored at −80 °C until use. Cytokines, chemokines and growth factors levels were evaluated using a Bio-Plex MAGPIX Multiplex Reader system (Bio-Rad, Milan, Italy) equipped with a Bio-Plex Manager software v 6.1 (BioRad) according to manufacturer’s instructions [58]. All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Measurements were performed in triplicate on samples (50 µL) using Bio-Plex Pro™ Human Cytokine 27-plex Assay (Bio-Rad). Standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software. Data were expressed as mean ± SD.

3.7. Bacterial Strains and Growth Conditions

CBDA was tested against Escherichia coli ATCC 13762, Staphylococcus aureus ATCC 6538P, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212. All bacterial strains were cultured on BD Brain Heart Infusion broth (BHI) (BD, Franklin Lakes, NJ, USA) or on BHI agar (OXOID, Basingstoke, Hampshire, England). The microorganisms were stored at −80 °C in BHI broth containing 10% glycerol (v/v) (Carlo Erba, Reagents, Milan, Italy) until use and the working cultures were activated in the respective broth at 37 °C with shaking at 225 rpm for 15–18 h. Overnight cultures were diluted to the required cell concentration (≈10⁶ CFU/mL) in BHI broth for growth experiments.

3.8. Biofilm Formation Assay

Biofilm formation assay of E. coli ATCC 13762, S. aureus ATCC 6538P, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212 was conducted in 96-well plates. Bacteria from overnight cultures were diluted and growth until 0.5 McFarland. The bacteria were diluted 1:100 and plated in each well containing 200 µL of the BHI broth. To evaluate the impact of the CBDA on biofilm formation, the medium was supplemented with different concentrations of cannabis extract equal to MIC, 2X MIC, 4X MIC and incubated overnight. At the end of the incubation, the medium was removed; the biofilms were washed twice with PBS and colored with crystal violet (1%) for 30 min, then resuspended in 200 µL of absolute ethanol. Optical density measurements were performed with a spectrophotometer (Enspire, Perkin Elmer, Winter Street, Waltham, MA, USA) at 595 nm. Bacteria incubated with medium alone were used as negative controls, while positive controls were bacteria incubated in medium supplemented with gentamicin 0.2 µg/mL. S. aureus and P. aeruginosa biofilm formation was assessed also through imaging. After medium was supplemented with different concentrations of CBDA (4.0, 8.0, 16.0 µg/mL for S. aureus and 8.0, 16.0, 32.0 µg/mL for P. aeruginosa) and incubated overnight, biofilms were stained with a FilmTracer LIVE/DEAD biofilm viability kit (Thermo Fisher Scientific, Third Avenue, Waltham, MA, USA) according to the manufacturer’s instructions. Images were acquired by Cell Discoverer 7, Zeiss. Each assay was performed in triplicate and all data were expressed as the mean ± SD of three individual experiments.

The authors thank the PON Project “EThoS (nutraceutical and cosmeceutical hemp seed”, and the Head-on project “Programma VALERE 2019” from the University of Campania “Luigi Vanvitelli”.