Extraction, purification and in vitro assessment of the antioxidant and anti-inflammatory activity of policosanols from non-psychoactive Cannabis sativa L.

2.2. Raw plant material

The wax material obtained by supercritical fluid extraction (SFE) with carbon dioxide (CO₂) of non-psychoactive C. sativa inflorescences (hemp, Kompolti variety) was provided by Exenia s.r.l. (Pinerolo, TO, Italy).

2.3. Isolation of PCs from hemp wax

2.4. HPLC-ELSD analysis of PCs

The HPLC-ELSD analyses of PCs in the purified mixture from hemp wax were performed on an Agilent Technologies (Waldbronn, Germany) modular model 1260 Infinity II system, with a vacuum degasser, a quaternary pump and a thermostated column compartment [2]. The separation of the compounds of interest was carried out on an Atlantis™ dC₁₈ column (150 × 3.0 mm, 3 μm, Waters, Milford, MA, USA). The mobile phase was composed of ACN (solvent A) and a mixture MTBE-MeOH 90:10 (v/v) (solvent B), under the following gradient: 0–1 min isocratic elution at 20% B; 1–16 min linear gradient from 20 to 45% B, which was held constant for 4 min; the column was finally brought to 20% B in 5 min. The flow-rate was 1.5 mL/min and the injection volume was 10 μL. The ELSD evaporator temperature was set at 35 °C, while nebulizer temperature was 30 °C. Nitrogen flow rate was set at 1.50 SLM. All the samples analyzed in this study were injected in duplicate.

2.5. Sample preparation for biological assays

To favor the solubilization of the PC purified mixture in the medium, a mixture of EtOH-DMSO (50:50, v/v) was used; then, Tween20 was added to reach a final concentration of 0.002%. The stock solution (20 mg/mL) was diluted with the cell culture medium to get the desired concentration range.

2.6. Cell culture and cell treatment

HaCaT keratinocytes were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM/F12 with 10% heat-inactivated FBS, l-glutamine 1% and Pen-Strep 1%. Cells were kept in a humidified incubator at 5% CO₂ and 37 °C until 80% of cell confluence. Once the confluence was reached, cells were treated with PCs (5–100 μg/mL) for 12 h and, subsequently, with 350 μM of H₂O₂ for 30 min. All the tests have been performed in serum-free medium.

2.7. Determination of reactive oxygen species content in HaCaT cells

The intracellular reactive oxygen species (ROS) level was evaluated using a commercial H₂DCF-DA kit (Abcam ab133851), containing a chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate, which is an oxidative stress indicator. In the presence of ROS inside cells, H₂DCF-DA is converted into dichlorofluorescein (DCF), a fluorescent adduct. Therefore, the fluorescence signal intensity of DCF is proportional to the amount of ROS produced by the cells.

To test the capacity of PCs to reduce ROS intracellular level in H₂O₂ stimulated keratinocytes, HaCaT cells were seeded in 96-well plates at a cell density of 2.5 × 10⁴ cells/well and cultured for 16 h before the experiment. Then, cells were treated with PCs (5, 12.5, 25, 50, 100 μg/mL) for 12 h. After this, the medium was removed and cells were rapidly washed with the buffer. Then, cells were stimulated for 20 min with 350 μM H₂O₂. After the stimulation, 100 μL of the diluted DCFDA solution was added to each well and plates were incubated for 45 min at 37 °C in the dark. Subsequently, the DCFDA solution was removed and 100 μL of buffer were added. NAC 10 mM was used as the positive control. Using the microplate reader VICTOR® Nivo™ 3s, (PerkinElmer Italia, Milan), fluorescence was recorded at λ_Ex/Em 488/535 nm, every 5 min for 1 h. All samples were analyzed in replicate (n = 6). ROS level produced by tested samples was reported as fold change in comparison with not treated cells (control), normalized as 1.

To examine the effect of the PC treatment on intracellular ROS production in H₂O₂-stimulated HaCaT cell line in different and integrative way, the fluorogenic probe CellROX® Green Flow Cytometry Assay Kit (Life Technologies, Milan, Italy) was used. Briefly, HaCaT derived keratinocytes cells were plated in μ-slide-8 well glass bottom (Ibidi, USA) at 6 × 10⁴ cells/well and kept in incubator overnight. Cells were then stimulated for 30 min with 350 μM H₂O₂ in the absence or presence of PC pre-treatment (100 μg/mL) or NAC (10 mM) (Sigma Aldrich. Milan). Subsequently, 10 μL of “CellROX Green” solution were added in each well 30 min before the end of the treatment at the concentration of 5 μM and fluorescence was measured by a 20 × fluorescence microscope Olympus IX 70 inverted microscope equipped with a Digital Sight camera DS-Qi2 (Olympus Life Science, Tokyo, Japan). Quantitative analysis was carried out using ImageJ (NIH) version 1.54e (USA) software. The results were expressed as a percentage of the control group.

2.8. Assessment of the antioxidant activity against catalase and superoxide dismutase

The antioxidant activity of the purified mixture of PCs was assessed by means of enzymatic assays based on their ability to remove H₂O₂ and the radical superoxide anion O₂^•–, which represent two of the most significant ROS species. In analogy with the role of the enzyme catalase (CAT) and superoxide dismutase (SOD), these were named CAT-like activity and SOD-like activity, respectively [14,15]. For each assay the sample was tested at 5, 12.5, 25, 50 and 100 μg/mL and NAC at 10 and 1 mM was used as a reference.

CAT-like activity tests were performed using the Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich) with a TECAN GeniosPro microplate reader. The presence of H₂O₂ was detected thanks to its reaction with a molecular probe catalyzed by the peroxidase enzyme, which generates a red fluorescent product that can be analyzed fluorometrically. CAT activity is reported as the percentage of H₂O₂ decomposed at the end of the assay. Twenty μL of each sample was treated with 200 μL of a 50 μM solution of H₂O₂ in buffer solution and measured the residual H₂O₂ concentration after 30, 60 and 120 min. Two replicates were performed for each sample.

SOD-like activity tests were carried out using the SOD determination kit (Sigma-Aldrich), adapted for a UV–Vis spectrophotometer (JASCO V-570). In this assay, the SOD-like activity is expressed as the inhibition rate (I.R.%) of the formation of a H₂O-soluble formazan dye, generated upon reduction of a tetrazolium salt (WST-1) by the superoxide anion catalyzed by xanthine oxidase and inhibited by SOD. This activity was evaluated after 20 min of treatment at 440 nm.

2.9. Immunoblotting

Western blot was used to measure the expression of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) in H₂O₂ induced ROS production in HaCaT cells pre-treated or not with PCs. Briefly, 350 μM H₂O₂ and PC (100 μg/mL) treated HaCaT cells were lysed and extracted in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% sodium deoxycolate, 1% Triton X-100, 2 mM PMSF). Protein quantification was carried out using BCA Protein Assay Kit (Life Technology, Milano, Italy), following the manufacturer instruction. Equal amounts of proteins (5 μg for each sample) were separated through 5–12% SDS-PAGE mini gel and transferred onto nitrocellulose membrane (Life Technology, Milano, Italy). The membranes were then incubated with primary rabbit antibody anti-MK2 (1:2000) (Cell Signaling Technology, USA) and anti–HO–1 (1:1000) (Abcam, UK) overnight at 4 °C. Then, the membranes were washed three times in TBST, incubated for 1 h with HRP-conjugated anti-rabbit antibody (Thermofisher, USA), and protein bands were observed and photographed. The densitometry analyses were performed using the Imagestudio lite software, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the loading control.

2.10. Evaluation of the anti-inflammatory activity of the purified mixture and cellular vitality

The in vitro anti-inflammatory activity of PCs from hemp wax was assessed using a validated cellular model previously described [16], where the inflammatory target nuclear factor kappa B (NF-κB) is monitored using a gene-reporting method in commercial R3/1-NF-κB cells. To verify that the presence of Tween20 did not affect the anti-inflammatory activity of the hemp wax, Tween20 at its highest concentration was also tested.

Briefly, R3/1-NF-κB cells were seeded (4000 cells/well) in a white 96-well plate (BRANDplates®, cell grade). Cells were pre-treated with different concentrations of hemp PCs (5–100 μg/mL) for 18 h in complete medium (DMEM 10% FBS, 1% l-glutamine, 1% Pen-Strep), followed by a 6 h stimulation with 10 ng/mL TNFα. Stachydrine (100 μM) was used as the positive control. To avoid component interference with the reading of the luciferase assay, cells were washed once with 100 μL of warm PBS and 50 μL of DMEM was then added. Next, 50 μL ONE-Glo™ Luciferase Assay Substrate (Promega Corporation, Madison, WI, USA) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, PerkinElmer™ Life Science, Monza, Italy).

The MTT assay was applied to assess cell viability for the all the concentrations tested in the anti-inflammatory assay. After 18 h incubation with hemp PCs (5–100 μg/mL) and Tween20 (0.002 %), the MTT reagent was added and incubated for 4 h. After medium removal, cells were lysed and MTT was solubilized by adding 100 μL of lysis buffer (8 mM HCl, 5% Tween20, DMSO). The 96-well plate was shaken for 15 min and the absorbance at 575 nm was measured using a plate reader (BioTek’s PowerWave HT, Winooski, VT, USA). Cells incubated with complete medium were used as a control for 100% cell proliferation.

2.11. Determination of the neutrophil elastase inhibitory activity

The inhibitory effect of neutrophil elastase was assessed using the Sigma-Aldrich kit Assay (Sigma-Aldrich, Milan, MAK213), which is based on the interaction between elastase and a synthetic substrate. The proteolytic cleavage releases a fluorophore which can be easily quantified by fluorescence. The test was performed according to the data sheet in the range of final concentration of tested sample 5–100 μg/mL. Enzyme activity and inhibition exerted by tested samples were calculated and IC₅₀ value extrapolated from the curve concentration/inhibition activity.

3.2. Qualitative and quantitative analysis of PCs in the purified mixture obtained from hemp wax

Quantification of PCs in the purified mixtures obtained from hemp wax was obtained by means of a validated HPLC-ELSD method [2]. Fig. 1 shows a representative HPLC-ELSD chromatogram of PCs from hemp wax. Resolution (Rs) was found to be higher than 1.50 for all peaks, except for peak 4.

Quantitative data related to the content of PCs in the product purified from hemp wax are shown in Table 1. Total PC amount wax was interestingly high with a value of 698.6 ± 9.5 mg/g, thus highlighting the role of this product in the pharmaceutical and nutraceutical fields. In the purified mixture the major compounds were found to be C₂₆OH and C₂₈OH. Together with PCs, other compounds are present in the final mixture and eluted at the beginning of the chromatographic run, mainly long-chain fatty acids that were not totally removed during the H₂O-washing process, as confirmed by the analysis using gas chromatography coupled with mass spectrometry (GC-MS) [13].

3.3. Effect of hemp PCs on the production of intracellular ROS

Stress is known to exert repercussions on several aspects of cell life. Various experimental research has demonstrated how intrinsic and extrinsic (smoking, UV exposure, environmental pollution) biological mediators of stress are able to induce aging in biological matrices and they can significantly promote cellular senescence, which is an important contributor to intrinsic and extrinsic skin aging [17]. Thus, among other factors, such as inhibition of telomerase activity and mitochondrial damage, oxidative damage plays a fundamental role [18,19]. Indeed, ROS produced through H₂O₂ is one of the trigger points to the molecular response of skin cells. In the light of this, the effect of acute exposure of HaCaT cells to 350 μM H₂O₂ was assessed. The choice of the exposure time and concentration was based on dose-response experiments at different time points (Fig. S2, Supplementary Information).

In the first test using DCF, PCs were able to efficiently counteract intracellular ROS upregulation mediated by H₂O₂. In detail, DCF fluorescence markedly increased in H₂O₂ stimulated cells already after 10 min of exposure (+20%, p < 0.01 vs ctrl), but at 100, 50 and 25 μg/mL PCs inhibited ROS production in stimulated cells in a clear extent, between 67.2 ± 10.2% and 76.9 ± 10.2% (at 100 μg/mL) compared to non-treated. Differences between values obtained for different concentrations were not statistically significant, even if compared with NAC as antioxidant reference compound (Fig. 2).

The ROS-sensitive fluorescent dye Green CellROX was also used to optically investigate whether PCs at the most effective concentration (100 μg/mL) may prevent H₂O₂-induced ROS generation. HaCaT cells exposed to 350 μM H₂O₂ for 30 min accumulated a markedly amount of intracellular ROS, as shown by the increase of fluorescence signal in comparison to the no treated cells (Fig. 3A). On the contrary, the pretreatment with PCs dramatically reduced the intensity fluorescent green signal at the level of NAC, indicating an inhibition of intracellular ROS production (Fig. 3A). As shown in Fig. 3B, the levels of ROS production elicited by H₂O₂ rise to 234.9 ± 6.1%, whereas the pretreatment with PCs reduced the level of ROS to 148.0 ± 8.5%, level close to that obtained with the treatment of 10 mM of NAC (115.0 ± 4.4%).

3.4. Antioxidant activity of the purified mixture of PCs against CAT and SOD

The ability of PCs to reduce the H₂O₂-induced ROS production led us to evaluate the possible activation of protein involved in H₂O₂ scavenger activity. The analysis of the antioxidant activity revealed that cells treated with PCs did not show a marked antioxidant activity against H₂O₂ and O₂^•–. In fact, the CAT-like activity test showed constant H₂O₂ concentration up to 120 min of contact with the samples. Analogously, the SOD-like activity test did not evidence any ability of the sample to scavenge O₂^•–, as the samples show no significant I.R.%. In the case of NAC, we observed around 50% dismutation of H₂O₂ connected with the CAT-like activity, while we did not detect any SOD-like activity.

These results suggest either an alternative pathway or others antioxidant response element (ARE) involving the activity of PCs on ROS modulation. Among them, HO-1, an enzyme regulated by ARE, plays a potentially key role in antioxidant defense and iron homeostasis [20]. In addition, several research identified signal transduction pathways involved in ROS regulation, such as the MK2/Heat shock protein 27 (Hsp27) signaling [21].

3.5. Effect of hemp PCs on MK2 and HO-1 expression in oxidative stress-induced HaCaT cells

The mitogen-activated protein kinase family (MAPK) is over-activated by external stressors and it plays a relevant role in immune, inflammatory, and apoptotic responses [22]. In particular, MK2, a member of MAPK, is known to be involved in oxidative stress [21]. Moreover, a relation between MK2 expression and HO-1 activity has been proposed in hepatobiliary cancer cells [21]. Therefore, the MK2 and HO-1 protein expression was assessed in this work in presence of H₂O₂ in HaCaT cells pretreated or not with PCs. Immunoblotting showed that 30 min of H₂O₂ treatment decreased the expression of HO-1, while MK2 expression remained unaltered in comparison to the control (Fig. 4A). However, the pretreatment with PCs resulted in a significant up regulation of both MK2 and HO-1 protein expression, when used in co-treatment with H₂O₂ in comparison to the H₂O₂ treatment alone (Fig. 4A). Indeed, as shown in Fig. 4B, the levels of the band intensity of MK2 range from 32.9 ± 18.0% in the cells treated with H₂O₂ to 88.7 ± 15.0% in the co-administration H₂O₂/PC pretreated cells. Similarly, the levels of HO-1 protein range from 123.0 ± 13.0% in the HaCaT treated with H₂O₂ to 171.0 ± 42.0% in the co-administration H₂O₂/PC pretreated cells (Fig. 4C). Interestingly, the levels of MK2 and HO-1 protein expression in the PC pretreated HaCaT cells were significantly greater than the control, reaching the 184.7 ± 18.0% and 219.0 ± 43.0%, respectively (Fig. 4B and C).

3.6. Anti-inflammatory activity of hemp PCs

Recently, it has been proposed a novel mechanism of inflammation, where the interaction of oxidative stimulus and inflammatory possess are tightly linked in a positive feed-back manner, which, starting from a non-clinical symptom might chronically lead to either a systemic or local damage [23]. Among the different pathways involved in oxinflammation, NF-κB plays a pivotal role [24]. Moreover, it is now well-established that MK2 is implicated in dampening the activity of NF-κB by regulating p38 [25]. Thus, we thought to evaluate its level upon treatment with PCs in the presence of an inflammatory stimulus. As described previously [26], the assay applied in this work evaluates the ability of either a molecule or a mixture to reduce the NF-κB activation induced by TNFα. The reduction of the luminescence signal is related to the reduction of NF-κB activation.

As highlighted in Fig. 5, PCs from hemp wax inhibited the upregulation of NF-κB induced by TNFα in a dose-dependent manner (even if at the concentrations 5, 12.5 and 25 μg/mL the reduction is not significant, a trend can be observed). In particular, at the highest concentration tested (100 μg/mL), there is a 1.7-fold reduction, which is not so far from the positive control (stachydrine 100 μM, 2.4-fold). The treatment with both PCs from hemp wax and Tween20 had no significant negative effects on cell viability (Fig. S4, Supplementary Information).

The authors are grateful to Dr. Alfonso Botto (Exenia s.r.l., Pinerolo, TO, Italy) for having provided the sample of hemp wax.