Rooted in therapeutics: comprehensive analyses of Cannabis sativa root extracts reveals potent antioxidant, anti-inflammatory, and bactericidal properties

2.2 Extractions

Three different types of extraction were performed: aqueous, alcohol, and acid-base. The proportion of solvent and roots used for each extract were neighboring 10 mL per gram of dried roots. All filtrations were performed by gravity using Whatman paper #5. To mimic traditional phytotherapy methods, the water extraction was conducted in the form of a decoction: the ground roots were macerated in distilled water for 24 h before being gently heated (75°C, 10 min) and filtered. The alcoholic extraction was carried out using continuous solvent extraction (Soxhlet) with ethanol (95%) (Thermo Fisher Scientific, Ottawa, Canada) for a period of 3 hours and 30 min. The acid-base extraction was performed following the method described by de Andrade et al. (2014), with some modifications. The root material was macerated for 24 h in methanol (Thermo Fisher Scientific, Ottawa, Canada) at room temperature and then filtered. The crude extract was acidified with 2% sulfuric acid (Thermo Fisher Scientific, Ottawa, Canada) to a pH of 2 and extracted with chloroform (Thermo Fisher Scientific, Ottawa, Canada) to remove neutral compounds. The aqueous acid solution was basified with 7 N ammonia (Thermo Fisher Scientific, Ottawa, Canada) to a pH of 10, then extracted with chloroform, with said chloroform subsequently evaporated in speedvac to obtain the acid-base extract. All extractions were reported as dry weight and then diluted in a 70% ethanol solution to a final concentration of 5 mg/mL, or in 20% ethanol, with the same concentration (5 mg/mL), for antibacterial and antifungal tests. Extraction yields are reported in the Supplementary Table A1).

2.3 Analysis of root extracts chemical composition

The UPLC‐QTOF‐MS analyses were carried out externally by the Centre de Recherche Industrielle du Québec (CRIQ). Briefly, a UPLC analysis was performed using a Waters Acquity Ultraperformance LC system (Waters), equipped with a binary pump system (Waters). An Acquity Ethylene Bridged Hybrid (BEH) C18 column (100 mm 2 mm id, 1.7 mm particle size) from Waters was used. The molecules of the Cannabis root ethanol extract were separated with a mobile phase that consisted of 0.2% acetic acid (eluent A) and acetonitrile (eluent B). The flow rate was 0.2 mL/min and the gradient elution was initial, 2% B; 0–1 min, 2%–100% B; 1–30 min, isocratic 100% B; 30–33 min, 100%–2% B; 33–33.5 min, isocratic 2% B; 33–40 min. The mass spectrometry (MS) analyses were carried out on a QTOF Micro mass spectrometer (Waters) equipped with a Z‐spray electrospray interface. Each analysis was performed in both positive and negative mode, and the data were acquired through a mass scan from 100 to 1,250 m/z. The ionization occurred at 120°C using a cone gas flow rate of 50 L/h, desolvation gas flow rate of 350 L/h, and a desolvation temperature set at 200°C. Nitrogen (99% purity) was used as a nebulizing gas. Data interpretation was carried out with the MassLynx 4.1 software. Mass extraction, deconvolution, and isotope and library search were performed using MZMine 2 (Pluskal et al., 2010).

2.4 Total phenolic content (TPC)

The TPC quantification was carried out using the Folin-Ciocalteu colorimetric method (Magalhães et al., 2010) with some modifications. In summary, 500 µL of the extracts were mixed with 250 µL of Folin-Ciocalteu reagent (Sigma-Aldrich, Darmstadt, Germany) and vortexed for 3 min. Subsequently, 500 µL of 8% Na₂CO₃ (ACROS organics from Fisher, Geel, Belgium) were added, and the volume was adjusted to 2.5 mL with H₂O. The mixture was incubated for 1 h in the dark at room temperature. Finally, the samples were centrifugated at 1,000 g for 5 min at room temperature, the supernatant was transferred to a microplate and absorbance was read at 765 nm on a spectrophotometer (Synergy H1, BioTek). The total polyphenol content was calculated as gallic acid (Thermo Fisher Scientific, Ottawa, Canada) equivalent (Supplementary Figure A1).

2.5 ABTS• radical scavenging activity

The assessment of the extracts’ antioxidant capacity was conducted using the method outlined by Re et al. (1999). In summary, the ABTS• radical (2.2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (diammonium salt) (Sigma-Aldrich, Darmstadt, Germany) was generated by oxidation with potassium persulfate (MAT laboratory, Québec, Canada) and can be reduced by a hydrogen-donor antioxidant. The reagent consists of a 7 mM ABTS and 2.45 mM potassium persulfate stock, kept at room temperature for 12 h in the dark. The reaction involved mixing 0.2 mL of sample with 2 mL of the ABTS• solution (diluted in ethanol). A₇₆₅ readings were taken at the beginning of the reaction and after 6 min (Synergy, BioTek). Trolox (6-hydroxy-2.5,7,8-tetramethylchromane-2-carboxylic acid) (Sigma-Aldrich, Darmstadt, Germany) was used to build standard curves at the start and end of the reaction and the results are expressed in Trolox equivalents (µM).

2.6 Ferric reducing antioxidant power (FRAP)

The ability of the extracts to reduce Fe³⁺ was demonstrated using the FRAP method (Bolanos de la Torre et al., 2015). In summary, a working solution was freshly prepared by mixing 10 volumes of acetate buffer (300 mM, pH 3.6) (Thermo Fisher Scientific, Ottawa, Canada) with one volume of 2,4,6-tripyridyl-s-triazine (TPTZ) (40 mM dissolved in 40 mM HCl) (Sigma-Aldrich, Darmstadt, Germany) and one volume of iron chloride (20 mM in water) (Thermo Fisher Scientific, Ottawa, Canada), warming to 37°C for 10 min before use. Gallic acid (1,000 µM) was employed as a standard. 20 μL of the extracts and the standard were dispensed into the wells of a 96-well microplate, followed by the addition of 280 µL of working solution. The mixture was agitated and incubated at 37°C in the dark for 30 min. Absorbance was measured at 593 nm (Synergy H1, BioTek).

2.7 Lipid peroxidation on linoleic acid micelles

Micelles were prepared as follows: 1 g of linoleic acid (ACROS organics from Fisher, Geel, Belgium) was dissolved in 222.72 mL of borate buffer (0.1 M, pH 9, SDS 0.1 M) (Thermo Fisher Scientific, Ottawa, Canada) purged with nitrogen beforehand. The mixture was incubated at 37°C with agitation for 10 min. To assess the appearance of conjugated dienes, 185 µL of 1X Phosphate-Buffered Saline (PBS) was mixed with 2 µL of micelles and 5 µL of sample. 18 μL of an 8 mM solution of 2.2′-Azobis (2-amidinopronane) dihydrochloride (AAPH) (Thermo Fisher Scientific, Ottawa, Canada) was added to each well just before the start of the absorbance reading on the microplate reader. Readings were taken every 5 min over a 5 h period at 234 nm (Synergy H1, BioTek). Ascorbic acid (Sigma-Aldrich, Darmstadt, Germany) was used as a positive control in the same concentration as the extract (5 mg/mL).

2.8 Lipid peroxidation of mitochondrial membranes

To measure the antioxidant capacity of the extract in a cellular context, THP-1 cells (American Type Culture Collection [ATCC], Manassas, VA, United States) were exposed to various extracts (1.25 μg/mL) for 24 h at concentration of 2–5 × 10⁵ Cells/mL in 96-wells plate. Subsequently, they were washed twice with PBS. Next, 100 µL of MitoPerOx at 100 nM (Cayman chemical company, Ann Arbor, United States) was added to the wells, followed by an incubation for 30 min. The cells were once again washed twice with PBS, and 500 µM hydrogen peroxide (H₂O₂) (Sigma-Aldrich, Darmstadt, Germany) was applied to the cells. After 1 h, the cells were analyzed on a flow cytometry (Cytoflex S, Beckman Coulter). The data was processed with the Flowjo software (v 10.9, BD Biosciences) and the 520/590 mean fluorescence ratio was calculated. This probe specifically binds to the mitochondrial membrane and emits maximum fluorescence at 590 nm. When the membrane is damaged, the emission maximum is shifted to 520 nm. An increase in the 520/590 nm ratio indicates mitochondrial lipid peroxidation.

2.9 Cholinesterase inhibition

To assess cholinesterase inhibition, the extracts and standards were diluted to a concentration of 0.125 μg/mL in phosphate buffer (0.1 M, pH 7.5) (Thermo Fisher Scientific, Ottawa, Canada). The reaction buffer consisted of 5.5′-Dithiobis-2-Nitrobenzoic Acid (DTNB) at 0.01 M and the enzyme at 100 U/mL in phosphate buffer at 0.1 M (75 µL per reaction) (Abcam, Waltham, United States). In each well, 75 µL of reaction buffer and 5 µL of sample were added, followed by a 5-min incubation at room temperature. Subsequently, 20 µL of acetylthiocholine iodide at 0.01 M (Abcam, Waltham, United States) were added, the plate was mixed, and absorbance was measured in a kinetic mode every 2 min at 410 nm. Inhibition was calculated using the following formula:

where Δi represents the absorbance difference between the two readings in the presence of the extracts and Δe is the corresponding difference for the solvent control.

2.10 Antibacterial and antifungal capacity

The extracts were tested in 96-well plates at a concentration of 5 mg/mL in 20% ethanol. 50 μL of a 0.5 McFarland unit suspension of the microorganisms to be tested (Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 6538 and Candida albicans) were added. The yeast strain was supplied by the microbiology laboratory of the Université du Québec in Trois-Rivières (Québec, Canada). The plate was incubated for 3 h for bacteria and 6 h for C. albicans. Then, 40 µL of p-iodonitrophenyltetrazolium (INT; 2.85 g/L) (Sigma-Aldrich, Darmstadt, Germany) were added to the wells, followed by an incubation of 1 h for bacteria and 16 h for C. albicans. The wells displaying a yellow color, indicating growth, were spread on agar plates [potato dextrose broth (PDB), potato dextrose agar (PDA), trypticase soy broth (TSB), and trypticase soy agar (TSA)] respectively; Thermo Fisher Scientific, Ottawa, Canada), and the bactericidal/fungicidal activity was evaluated based on the number of colonies observed.

2.11 IL-6 production

THP-1 cells were cultivated in complete Roswell Park Memorial Institute (RPMI) medium (Wisent Inc., St-Bruno, Canada) complemented with 10% fetal bovine serum (Wisent Inc., St-Bruno, Canada) and 100 I.U./ml penicillin/streptomycin (Sigma-Aldrich, Darmstadt, Germany) which was refreshed every 2–3 days. The cells were transferred into plates and differentiated into macrophages with 200 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Darmstadt, Germany) for 24 h. After differentiation, PMA was removed, and the extracts and control were added for a 24 h period. Positive control used was zingerone, a known anti-inflammatory agent, in concentrations of 50 μM. The differentiated THP-1 were stimulated with E. coli 0111: B4 lipopolysaccharide (LPS) (Sigma-Aldrich, Darmstadt, Germany) at a concentration of 500 ng/mL for 24 h. The supernatants were collected for enzyme-linked immunosorbent assay (ELISA) for interleukine-6 (IL-6) in accordance with the manufacturer’s protocol (CAT #555220, BD Bioscience, Heidelberg, Germany).

2.12 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from THP-1 cells using TRIzol reagent (Invitrogen, Carlsbad, United States) and subjected to reverse transcription with M-MLV reverse transcriptase (NEB, Whitby, Ontario) following the manufacturer’s instructions. The relative mRNA expression levels were quantified using the Luna Universal qPCR kit (NEB, Whitby, Ontario). All samples were analyzed in triplicate and normalized using β-actin as the reference gene. The list of primer sequences is available in Table 1.

3.2 Total polyphenol content and antioxidant capacity

Phenolic compounds such as polyphenols are vital plant specialized metabolites known for their protective role against UV radiation and potential in reducing the risk of age-related diseases. In this study, we analysed extracts derived from the roots of plants cultivated hydroponically (CH) and in soil (CS), all originating from the same mother plant. The extracts were obtained using water (H₂O), ethanol (EtOH), and an acid-base protocol (AB). The quantity of phenolic compounds present in the six extracts was determined using the Folin-Ciocalteu method (Figure 1A). Notably, ethanol extractions exhibited the highest concentration of polyphenols, followed by water and AB extractions, irrespective of the cultivation method. Furthermore, extracts from soil-cultivated plants generally displayed slightly elevated concentrations of phenolic compounds with increases of 54% for alcohol extraction, 30% for water extraction, and 35% for acid-base extraction compared to hydroponic cultivation (Figure 1A).

To assess the antioxidant capacity of the extracts, the ABTS radical neutralization assay was employed, measured in Trolox equivalent (µM). Results indicated that CS-EtOH and CH-H₂O extracts exhibited the highest neutralization capacity, while the acid-base extracts demonstrated the lowest efficacy, correlating with their total polyphenol profiles (Figure 1B). No significant neutralization capacity was observed in the solvent-only controls. Additionally, the antioxidant capacity, through the ferrous ion-reducing capacity assay, calculated in gallic acid equivalent (µM), was evaluated (Figure 1C). It is interesting to note that the pattern observed mirrored that of the ABTS test, with ethanolic extracts and aqueous extracts displaying superior antioxidant capacity compared to the acid-base extractions. These findings suggest a pivotal role of phenolic compounds in determining the antioxidant capacity of the extracts.

3.3 Lipid peroxidation inhibitory activity

Next, we assessed the membrane protection provided by the various extracts using linoleic acid micelles. When added to these micelles, the oxidant AAPH initiates a chain reaction of lipid peroxidation that can be tracked by the appearance of conjugated diene systems, which have a maximum absorbance at 234 nm. Protection against lipoperoxidation, or the decrease in the formation of conjugated diene systems, is correlated to the antioxidant capacity of an extract. The AB extracts did not prevent lipid peroxidation, while the EtOH extracts were the most inhibitory and the H₂O extracts showed intermediate activity for both hydroponic and soil cultivation (Figures 1D, E). Most extracts, as well as the ascorbic acid positive control, progressively lost their effectiveness over time. In contrast, the CS-EtOH extract maintained a relatively stable protection after 5 h, with an average absorbance inhibition of 60%. The observed reaction kinetics differences between the extracts and ascorbic acid control suggests that they act through different molecular antioxidant mechanisms. As was observed with the two previous antioxidant assays, the profile of lipid peroxidation inhibition of the extracts seems to follow their polyphenol profiles.

Protection against in-cell oxidative stress was measured using the MitoPerOx probe fluorescence before and after the treatment of THP-1 cells with H₂O₂ in the presence or absence of the extracts. As expected, the AB extracts from both hydroponic and soil-cultivated plants had non-significant antioxidant effect, as surprisingly did CH-H₂O and CS-EtOH, while CH-EtOH and CS-H₂O extracts significantly reduced the impact of oxidative stress on mitochondrial membranes by 86% and 72%, respectively, relative to the solvent control (Ctrl) (Figure 1F). Interestingly, the CH-EtOH extract offered slightly superior protection when compared to the Trolox control (10 µM).

Overall, significant antioxidant activity was observed in aqueous and ethanolic extracts from both soil and hydroponic cultivated plants, whereas acid-base extracts appeared to have limited to no effect. For the TPC, ABTS, and lipid peroxidation tests, soil extracts proved to be more potent, while for the FRAP test, hydroponic extracts exhibited higher activities. Intriguingly, the most promising extracts in the FRAP test (CH-EtOH and CS-H₂O) coincided with those providing significant protection to mitochondria, as evidenced by results obtained with the MitoPerOx probe.

3.4 Anti-inflammatory effect

The anti-inflammatory properties of the extracts were evaluated by measuring IL-6 production after LPS stimulation of differentiated THP-1 cells. Our results revealed a significant decrease in IL-6 concentration in the supernatant of cells treated with CS-AB and CS-H₂O extracts compared to the control, while there was no significant changed upon treatment with CS-EtOH (Figure 2A). In contrast, extracts from hydroponic culture did not demonstrate any anti-inflammatory effect, with the CH-EtOH extract even exhibiting a significant pro-inflammatory effect under the experimental conditions used. To ensure that the treatments did not compromise the cell metabolic activity, we performed an MTT viability assay (Supplementary Figure A2).

One of the pathways implicated in modulating the inflammatory response of THP-1 cells involves the Nrf2 transcription factor. This pathway, vital for inducing cytoprotective gene expression, serves as the primary defence against oxidative stress (Baird and Yamamoto, 2020). Activation of Nrf2 prompts its translocation into the nucleus, where it regulates the expression of various genes, including NAD(P)H quinone dehydrogenase (NQO1), cyclooxygenase 2 (COX-2), heme oxygenase (HO1), and inducible nitric oxide synthase (iNOS) (Saha et al., 2020). Subsequently, RNA extraction followed by qRT-PCR was performed to assess the modulation of Nrf2-dependent gene expression on the samples that had affected IL-6 levels (CS-AB, CS-H₂O and CH-EtOH) (Figures 2B, C).

It is noteworthy that few significant changes were observed in gene expression levels in response to treatment to Cannabis root extracts (Figures 2B, C). Specifically, the CS-H₂O extract, which reduced IL-6 production, elicited a decrease in NQ O -1 expression, a gene whose product is involved in the neutralization of reactive oxygen species. Conversely, the CH-EtOH extract, which increased IL-6 production, upregulated COX-2 expression, suggesting that its pro-inflammatory effect might arise from enhanced production of prostaglandin H2 (PGH2), a COX-2 activity product and precursor to biologically active prostanoids such as various prostaglandins and thromboxane A₂ (Olszowski et al., 2015). No significant difference in relative expression was detected for H O -1 and iNOS (Supplementary Figure A3).

3.5 Cholinesterase inhibitory activity

Cannabis roots contain poorly characterized nitrogen-containing compounds including alkaloids, such as cannabisativine (Table 2). Galanthamine, a prominent alkaloid predominately isolated from plants within the Amaryllidaceae family (Harvey, 1995), is renowned for its role in Alzheimer’s disease therapy through the inhibition of cholinesterase enzymes (Sharma, 2019). To expand our understanding of the pharmacological potential of Cannabis roots, we investigated this activity across various extracts.

The potential cholinesterase inhibition of Cannabis root extracts was evaluated using the catalytic reaction of acetylthiocholine iodide by the enzymes acetylcholinesterase and butyrylcholinesterase, with galanthamine serving as a positive control. Regarding the acetylcholinesterase inhibition, five out of the six extracts demonstrated stronger effects (ranging from 45% to 85%) than that of galanthamine (Figure 3). These results suggest that molecules with anticholinesterase effect are present in the extracts, but in varying proportions, and with different affinities for the two enzymes tested. Conversely, for the butyrylcholinesterase, all the extracts displayed a moderate inhibitory activity (ranging from 8% to 20%), akin to that of galanthamine (18%), with extracts from soil cultivation exhibiting greater inhibitory effects compared to those from hydroponic cultivation (Figure 3).

The authors would like to thank all the lab members for their technical support and useful advice. Authors also wish to thank the Centre de Recherche Industrielle du Québec (CRIQ), and particularly Pascal Dubé for UPLC-QTOF-MS analyses. Also, thanks are extended to the team at Innofibre for their contribution and collaboration with the antimicrobial testing. Warms thanks to Mélodie B. Plourde and Karen Cristine Goncalves dos Santos for proofreading this manuscript.