Inflammation is a protective response triggered by harmful stimuli such as infections or tissue damage [1]. Research has consistently demonstrated a strong and intricate link between oxidative stress and inflammation, considering the former as a primary cause of the latter [2]. Conventional treatments for these conditions often carry adverse side effects, highlighting the urgent need for alternative therapeutic approaches. The plant kingdom offers a vast array of compounds with potent antioxidant properties, and recent studies have revealed their effectiveness in regulating inflammation [3].
Cannabis sativa L., one of humanity’s oldest cultivated plants, has a long history in medicine [4], and has garnered substantial attention due to its increasing legalization worldwide [5]. For instance, Morocco has made significant strides in cannabis regulation. Law No. 13.21, enacted in 2021, legalized the cultivation, processing, and sale of cannabis for scientific and medical purposes [6]. The plant’s diverse chemical composition, which includes over 565 identified compounds such as cannabinoids, terpenes, and flavonoids, underpins its therapeutic potential [7,8,9]. The chemical profile (“chemotype”) of Cannabis sativa plants is used to classify Cannabis into different chemical varieties, or “chemovars.” These chemovars are distinguished by varying cannabinoid and terpene ratios, which are believed to produce unique effects in human consumers [10,11]. Delta-9-tetrahydrocannabinol (THC) is the primary compound responsible for the plant’s psychoactive effects [12]. In addition to THC, cannabis contains other cannabinoids without psychoactive effects such as cannabidiol (CBD) [13], one of the cannabinoids holding an important therapeutic potential among the multitude of compounds with null psychoactive properties [14]. Previous research has shown that components of cannabis extracts can effectively reduce inflammation in laboratory animal and human studies [15].
The aim of this study is to comprehensively characterize the phytochemical composition of hexanic and chloroformic Cannabis sativa L. extracts using high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and gas chromatography-mass spectrometry (GC-MS). The antioxidant and anti-inflammatory potential of these extracts will be evaluated through in vitro assays. Additionally, molecular docking studies being the prestigious tool to explore various interactions between protein and ligand [16,17] were used to explore the potential molecular targets and mechanisms of action underlying the observed bioactivities.
The Cannabis sativa L. plants used in our study were collected in September 2021, during the flowering phase, from Meadow Rif Mountain in the Tafrant region of Taounate Province, Morocco (34°39′28.4″ N, 5°05′58.9″ W). The climate is predominantly mountainous, characterized by cold, rainy winters and mild to hot summers. Average annual rainfall ranges from 700 to 1,300 mm. A dry spell typically occurs during the summer months (July to mid-August), with minimal rainfall and temperatures that can often reach or exceed 40°C. The plant material was sourced from local farmers who used seeds provided by the National Agency for the Regulation of Activities Related to Cannabis (ANRAC). A taxonomist from the Scientific Institute of Rabat, Morocco, verified the species to confirm its identity. A voucher specimen with the reference number RAB 112735 has been deposited in the institute’s herbarium for future taxonomic reference. The collected plant material was dried in a dark, well-ventilated area at room temperature (25 ± 2°C) until a constant weight was achieved. To decarboxylate acidic cannabinoids, such as tetrahydrocannabinolic acid and cannabidiolic acid, the dried plant matter underwent a 30-min heat treatment at 120°C, following the protocol established by Veress et al. [18]. Once dried, the plant material was finely ground using a laboratory-grade mill to ensure uniform particle size, promoting consistent extraction efficiency in subsequent analyses.
Twenty grams of dried plant material were subjected to ultrasound-assisted extraction (UAE) using 200 mL of chloroform and 200 mL of hexane, individually. The extraction process was conducted in an ultrasonic bath at 120 Hz and 30°C for 60 min. The resulting mixtures were filtered under vacuum to isolate the extracts. Solvent removal was achieved using a rotary evaporator at 40°C, yielding the chloroformic extract (CHL-CBD, enriched in CBD) and the hexanic extract (HEX-THC, enriched in THC), respectively. This extraction procedure was repeated three times for each solvent. The combined crude extracts demonstrated extraction yields of 5.67 ± 0.7% for CHL-CBD and 3.8 ± 0.24% for HEX-THC. Extracts were stored at −20°C until further analysis.
The TPC of the dried extracts was quantified using the Folin-Ciocalteu spectrophotometric method. Absorbance measurements were recorded at 750 nm using a spectrophotometer (Jenway 6305). TPC values were expressed as milligrams of gallic acid equivalents per gram of dried weight of extract (mg GAE/g DWE) and represent the mean value of three separate analyses [19].
Total flavonoid levels in the extracts were quantified using a spectrophotometric aluminum chloride colorimetric assay adapted from Ousaaid et al. [20]. Absorbance readings were taken at 510 nm. Results were expressed as milligrams of Quercetin equivalents per gram of dry extract (mg QE/g DW) and represent the average of three independent determinations.
Flavonol content (FC) was quantified using a modified spectrophotometric method adapted from Al-Dabbas et al. [21]. Quercetin served as the standard for constructing a calibration curve. Plant extracts (1 mg/mL) were reacted with 2 mL of aluminum chloride (2%) and 6 mL of sodium acetate solution (5%). The resulting mixture was incubated at 20°C for 2.5 h before absorbance measurement at 440 nm. FC was calculated from the calibration curve and expressed as quercetin equivalents per gram of dry extract (mg QE/g DW). The analysis was performed in triplicate.
Condensed tannin content was quantified using a modified spectrophotometric method adapted from Remok et al. [22]. Catechin served as the standard for calculating results, which were expressed as milligrams of catechin equivalents per gram of dry extract (mg CE/g DW). The experiments were performed in triplicate.
GC-MS analysis of hexane and chloroform extracts obtained from Cannabis sativa L. was performed using a Thermo Scientific TRACE GC ULTRA system [23]. The system was equipped with a split injector and coupled to a mass spectrometer. Separation of compounds was achieved on a TG-1MS capillary column (30 m × 0.25 mm, film thickness: 0.25 μm). Helium was used as the carrier gas at a constant flow rate of 1.5 mL/min. The GC oven temperature was programmed to increase from 90 to 250°C at a rate of 1°C/min, held for 3 min. Both the injector and interface temperatures were maintained at 250°C. Sample preparation involved a ten-fold dilution in cyclohexane, with 1 μL injected for analysis. Electron ionization at 70 eV was employed for mass spectral data acquisition in the m/z range of 50–550. Compound identification was accomplished by comparison with the NIST spectral library.
Sample and standard solutions were prepared at a concentration of 30 mg/mL and subsequently filtered through 0.4 µm membrane filters to remove particulate matter. Chromatographic analysis was conducted using a HPLC-DAD, following a modified method adapted from Zefzoufi et al. [24]. Separation was achieved on a Kinetex C18 reversed-phase column (250 × 4.6 mm, 2.6 µm particle size). A binary gradient elution was employed using a mobile phase consisting of 0.1% acetic acid in water (solvent A) and methanol (solvent B). The gradient profile was as follows: 0–3 min, 5–25% B; 3–6 min, isocratic at 25% B; 6–9 min, 25–37% B; 9–13 min, isocratic at 37% B; 13–18 min, 37–54% B; 18–22 min, isocratic at 54% B; 22–26 min, 54–95% B; 26–29 min, isocratic at 95% B; 29–35 min, 95–5% B; 35–45 min, isocratic at 5% B. The chromatographic system was operated at a flow rate of 1 mL/min with the column temperature maintained at 30°C. UV-Vis detection was performed within a spectral range of 200–400 nm, with chromatographic profiles monitored at 280 nm. Compound identification was based on retention time comparison with authentic standards.
The antioxidant capacity of the extracts was evaluated using a combination of spectrophotometric assays:
The antioxidant capacity of the cannabis extracts was assessed using the DPPH radical scavenging assay adapted from El Menyiy et al. [25]. The ability of the extracts to scavenge the DPPH radical was determined by measuring the decrease in absorbance at 517 nm and calculating the IC50 value. Butylated hydroxytoluene (BHT) was used as a positive control for comparison. Results were expressed as mean value ± standard deviation (SD) of three independent experiments.
The TAC of both extracts was assessed using the phosphomolybdenum assay, adapted from the protocol described by Laaroussi et al. [26]. The test was carried out in triplicate. Results were expressed as micrograms of ascorbic acid equivalents per gram of dry weight of extract (μg AAE/g DWE).
The antioxidant potential of the cannabis extracts was evaluated using the ABTS radical scavenging assay, adapted from Zouhri [27]. The extract’s ability to decolorize the ABTS radical cation was determined spectrophotometrically by measuring absorbance at 734 nm. Ascorbic acid served as a reference standard. The analysis was performed in triplicate.
The reductive potential of the extracts were assessed using the FRAP assay [28]. Absorbance was measured at 700 nm to determine the IC50 value. The test was conducted in triplicate, with ascorbic acid serving as the positive control.
The in vitro anti-inflammatory potential of the extracts was assessed using the albumin denaturation assay adapted from the literature [27]. Briefly, 0.5 mL of bovine serum albumin (BSA) solution (0.2% w/v in Tris buffer, pH 6.8) was incubated with various concentrations of the plant extract or diclofenac sodium (positive control) for 15 min at 37°C. Subsequently, the mixtures were exposed to 72°C for 5 min to induce protein denaturation. The absorbance of the resulting solutions was measured at 660 nm to quantify protein aggregation. All experiments were performed in triplicate.
RBCs were selected as a model for membrane stabilization testing due to the similarity of their membranes to lysosomal membranes. Stabilizing the erythrocyte membrane also stabilizes the lysosomal membrane, which is crucial for limiting inflammation by preventing the release of harmful enzymes and proteases from activated neutrophils. This release can lead to inflammation and tissue damage [29,30].
A buffer solution was created by dissolving 100 g of sodium chloride (NaCl), 2.5 g of potassium chloride (KCl), 18 g of disodium hydrogen phosphate dihydrate (Na₂HPO₄·2 H₂O), and 8 g of potassium dihydrogen phosphate (KH₂PO₄) in 1 L of distilled water.
A 0.9% (w/v) saline solution was prepared by dissolving 9 g of sodium chloride in distilled water to a final volume of 1 L.
Blood samples (7 mL) were obtained from healthy human volunteers aged 20–30 years with no history of anti-inflammatory medication use in the month prior to the experiments. The samples were collected in heparinized tubes and subjected to centrifugation at 2,500 rpm for 5 min to separate erythrocytes. The erythrocyte pellet was washed three times with sterile saline (0.9% w/v NaCl) and subsequently resuspended in phosphate-buffered saline (pH 7.4) to prepare a 40% hematocrit suspension [31].
Heat-induced hemolysis was assessed using the method described by Gunathilake et al. [32]. In this procedure, 0.05 mL of a blood cell suspension was combined with 0.05 mL of various concentrations of extracts and 2.95 mL of phosphate buffer (pH 7.4) in triplicate. The resulting mixture was incubated at 54°C for 20 min in a shaking water bath. Post-incubation, the mixture was centrifuged at 2,500 rpm for 3 min. The absorbance of the supernatant was then measured at 540 nm using a UV/VIS spectrometer. A phosphate buffer solution served as the control in this experiment. Aspirin was used as the standard.
Hemolysis inhibition was calculated using the formula
In this assay, the method described by Aidoo et al. [33] was followed with slight modification. The test samples consisted of 0.5 mL of human red blood cell stock mixed with 4.5 mL of hypotonic solution (0.9% NaCl) containing varying concentrations of extracts. The negative control sample consisted of 0.5 mL of RBC suspension mixed with 4.5 mL of hypotonic solution alone. Indomethacin was used as the standard. The experiment was conducted in triplicates at each concentration. The mixture was incubated for 10 min at room temperature and then centrifuged for 10 min at 3,000 rpm. The hemoglobin content of the supernatant was measured using a spectrophotometer at 540 nm. The percentage inhibition of hemolysis was calculated using the following formula:
The crystallographic structure of the proteins of interest was obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/). The receptors represented by PDB IDs: 5LDE (The Nuclear Factor Kappa B [NF-kB] enzyme), 1EQG (Cyclooxygenase enzyme 1), 1CX2 (Cyclooxygenase enzyme 2), and 3V99 (lipoxygenase) were prepared by adding hydrogen atoms, Kollman charges, and removing irrelevant solvent molecules and co-crystallized ligands for virtual screening employing AutoDock Vina [34]. In this study, Chem3D was utilized to optimize the 3D structures of the ligands through energy minimization, ensuring their stability and accuracy [35,36] followed by saving these files in SDF format which were then converted to pdbqt format via Open Babel GUI software to prepare them for virtual screening using Auto Dock Vina. These structures were further refined to “pdbqt” format via AutoDock Tools-1.5.7. Molecular docking was executed with AutoDockTools-1.5.7, using grid box specifications determined through Discovery Studio, enabling the prediction of ligand binding within the active sites [37]. The process of virtual screening was conducted by employing the script-based approach of AutoDock Vina. The value for exhaustiveness was configured as 08, while the number of nodes was specified as 09. Docking scores highlighted interaction strengths, with more negative values presenting stronger ligand–protein affinities. Visual representation of ligand–protein interactions was executed via Discovery Studio 2021 Client, to explore the binding orientations.
Data were expressed as mean value ± standard deviation (SD). Statistical comparisons among groups were conducted using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparisons. Statistical significance was considered at p < 0.05, using GraphPad Prism software (version 9).
The phenolic composition of Cannabis sativa extracts was analyzed spectrophotometrically, and the results are summarized in Table 1. Spectrophotometric analysis revealed higher levels of flavonoids, flavonols, and total phenols in the hexane extract compared to the chloroform extract of Cannabis sativa. Conversely, the chloroform extract contained a greater amount of total tannins. These findings align with previous research [38,39]. The phenolic and flavonoid contents were lower than that reported in Moroccan cannabis seeds [40] but higher than those reported in the inflorescences and leaves of various industrial cultivars [41,42]. The antioxidant properties attributed to phenolics are due to their hydroxyl groups, which can inhibit lipid oxidation through chain-breaking mechanisms [43]. Furthermore, phenolic compounds suppress inflammation by inhibiting enzymes involved in prostaglandin and leukotriene production, such as phospholipase A2, cyclooxygenase, and lipoxygenase [44,45].
Content of TPCs, TFCs, FC and TTCs of Cannabis extracts
| TPC (mg GAE/g DWE) | TFC (mg QE/g DW) | FC (mg QE/g DW) | TTC (mg CE/g DW) | |
|---|---|---|---|---|
| CHL-CBD | 130 ± 2.5a | 6 ± 1.2a | 0.9 ± 0.2a | 0.46 ± 0.09a |
| HEX-THC | 175 ± 4b | 14 ± 1.5b | 2.2 ± 0.4b | 0.51 ± 0.08a |
Data are presented as mean value ± standard deviation. Values sharing the same letter within a column are not significantly different (p > 0.05).
To selectively target THC and CBD, chloroform and hexane were employed for extraction, respectively. The hexane extract exhibited a higher THC content (89.87%) compared to the chloroform extract, while the latter was enriched in CBD (47.38%), as determined by GC-MS analysis (Table 2, Figures 1 and 2). Previous studies have attributed diverse biological properties, including antioxidant and anti-inflammatory effects, to both cannabinoids (CBD, THC) [46,47,48,49,50,51]. Notably, in vivo research has demonstrated the predominantly anti-inflammatory effects of CBD, and CBD:THC combinations, in contrast to the neutral influence of THC on pro- and anti-inflammatory cytokine levels [52].
Cannabinoid profile of Cannabis sativa L. extracts and their percentage area
| Molecular weight | Molecular structure | Retention time | Area % | ||
|---|---|---|---|---|---|
| HEX-THC (%) | CHL -CBD (%) | ||||
| CBD | 314 |
| 22.86 | <1 | 47.38 |
| THC | 314 |
| 23.91 | 89.87 | <1 |
GC-MS chromatogram of chloroformic extract Cannabis sativa L. enriched in CBD.
GC-MS chromatogram of hexanic extract of Cannabis sativa L. enriched in THC.
HPLC-DAD analysis enabled the identification of four phenolic acids (
Identified compounds in cannabis sativa L. HEX-THC and CHL-CBD extracts using HPLC-DAD
| Pick number | Retention time | Compound | Area % | |
|---|---|---|---|---|
| HEX-THC | CHL-CBD | |||
| 1 | 3.35 |
| 2.2 | 0.5 |
| 2 | 5.45 | Gallic acid | 2.1 | 1.9 |
| 3 | 8.50 | Catechin hydrate | 1 | 0.8 |
| 4 | 14.68 | P-coumaric acid | 4 | 7.2 |
| 5 | 30.38 | Quercetin | 30 | 29.1 |
| 6 | 31.60 | Rosmarinic acid | 0.1 | 0.4 |
HPLC-DAD chromatogram of chloroformic extract of Cannabis sativa L. enriched in CBD.
HPLC-DAD chromatogram of hexanic extract of Cannabis sativa L. enriched in THC.
The identified phytoconstituents align with previous studies on Cannabis sativa. Izzo et al. [41] reported p-coumaric acid, catechin, and quercetin presence in industrial variety inflorescences. A comprehensive analysis by Jin et al. [54,55] revealed seven flavonoids in different plant parts. André et al. [42] reported similar findings. Gallic and rosmarinic acids were predominantly found in aerial parts [56], while p-coumaric acid and ethyl p-coumarate were identified as primary phenolics in hemp roots [57] (Figure 5).
The structure of identified compounds in HEX-THC and CHL-CBD extracts: (a)
Antioxidant capacity quantifies a biological system’s resistance to oxidative damage inflicted by reactive oxygen species. Given the diverse mechanisms underlying antioxidant activity, a single assay cannot comprehensively evaluate the antioxidant potential of a complex mixture. Hydrogen atom transfer and single electron transfer are the primary mechanisms underpinning antioxidant assays.
The antioxidant properties of both extracts were assessed using DPPH, FRAP, TAC, and ABTS assays (Table 4). Both extracts demonstrated significant antioxidant activity. As anticipated, the hexane extract, exhibiting the highest levels of polyphenols, flavonoids, and flavonols, showed the strongest antioxidant potential.
Antioxidant potential of Cannabis sativa L. extracts was assessed using DPPH, ABTS, TAC and FRAP assays
| DPPH (IC50 μg/mL) | ABTS (IC50 μg/mL) | TAC μg AAE/g DWE | FRAP (EC50 μg/mL) | |
|---|---|---|---|---|
| CHL-CBD | 45 ± 2.7a | 65 ± 5a | 88 ± 3a | 33 ± 2a |
| HEX-THC | 32 ± 43b | 47 ± 4.1b | 108 ± 4b | 26 ± 1.9b |
| BHT | 14.1 ± 1c | — | — | — |
| Ascorbic acid | — | 12.34c | — | 55.4c |
Values are expressed as mean value ± standard deviation. Different letters within a row indicate statistically significant differences (p < 0.05), n = 3.
DPPH IC50 values were 32 ± 43 μg/mL and 45 ± 2.7 μg/mL for hexane and chloroform extracts, respectively, surpassing the standard BHT (IC50 = 14.1 ± 1 μg/mL). ABTS results corroborated these findings, with IC50 values of 47 ± 4.1 μg/mL and 65 ± 5 μg/mL for hexane and chloroform extracts, respectively, significantly exceeding ascorbic acid (IC50 = 12.34 μg/mL). The FRAP assay, measuring ferric ion reduction to ferrous ions, indicated a higher antioxidant capacity in the hexane extract (EC50 = 26 ± 1.9 μg/mL) compared to the chloroform extract. However, both extracts exhibited lower reducing power than ascorbic acid (EC50 = 13.4 μg/mL). TAC values were 88 ± 3 and 108 ± 4 μg AAE/g DWE for hexane and chloroform extracts, respectively.
The results of the antioxidant tests reveal interesting properties, which align with previous research [42,56,58,59]. However, our findings differ from those of Hacke et al., who reported higher IC50 values of 202.8 ± 7.8 μg/mL for a THC-rich extract and 147.3 ± 18.7 μg/mL for a CBD-rich extract. Their study also demonstrated that pure CBD exhibited stronger antioxidant activity than pure THC in the DPPH assay, while THC showed greater antioxidant potential in the ABTS test. Additionally, they found that the 10:90 (CBD/THC) combination exhibited potent ABTS scavenging activity, whereas the 75:25 (CBD/THC) combination was the most effective in the DPPH test [60]. Hayakawa et al. also proved that CBD exhibited stronger antioxidative power than THC in an in vitro study using the DPPH radical [61]. In contrast, Borges et al. suggested that THC exhibits a slightly higher antioxidant potential than CBD [62,63].
The observed antioxidant activity likely originates from the synergistic interplay of THC, CBD, and phenolic compounds within the cannabis extracts [64]. Beyond their pharmacological effects, phenolic compounds, exemplified by quercetin, can enhance the bioavailability of other compounds [65], contributing to the extracts’ overall antioxidant capacity.
Inflammation is a protective response initiated in tissues to eliminate harmful pathogens or initiate tissue repair following injury or infection [66]. Protein denaturation is closely linked to inflammation and contributes to various inflammatory conditions [67]. Consequently, a substance’s ability to inhibit protein denaturation implies potential anti-inflammatory effects. The present study evaluated the in vitro protein denaturation inhibitory effects of two Cannabis sativa L. extracts. Figure 6 depicts the IC50 values obtained for BSA denaturation inhibition, The CHL-CBD extract displayed slightly lower activity (IC50 = 365.1 ± 5.5 μg/mL) compared to the HEX-THC extract (IC50 = 354.7 ± 4.5 μg/mL), with no significant difference. Diclofenac sodium exhibited the most potent protein protection (IC50 = 288 μg/mL), significantly outperforming both extracts (p < 0.01). Haddou et al. found that various extracts from Moroccan Cannabis sativa seeds demonstrated moderate inhibition of BSA denaturation [68].
IC50 values for inhibiting albumin denaturation, data are expressed as mean value ± SD, n = 3, **p < 0.01.
The remarkable inhibition of BSA denaturation of the HEX-THC, CHL-CBD demonstrating the potential anti-inflammatory properties of the plant may be linked to the high concentrations of cannabinoids, flavonoids, flavonols, phenols, and tannins in these extracts. Quercetin and p-coumaric acid were identified as major compounds contributing to BSA stability [69,70]. Cuinica and Chissico reported that quercetin possesses protective effect on protein denaturation but lower than that of hydroethanolic extract of Urtica dioica [71], suggesting that the anti-inflammatory action of the plant extracts might arise from a synergistic interaction among multiple constituents rather than a single compound.
Figures 7 and 8 illustrate a dose-dependent protective effect of both extracts against hemolysis.
Effect of HEX-THC, CHL-CBD, and aspirin on inhibition of hemolysis in heat induced test.
Effect of HEX-THC, CHL-CBD, and indomethacin on inhibition of hemolysis hypotonicity-induced test.
The hexane extract exhibited superior efficacy, with IC50 values of 185.94 ± 8.81 μg/mL and 264.51 ± 41.18 μg/mL for heat- and hypotonic-induced hemolysis, respectively, compared to the chloroform extract (IC50 = 301.55 ± 8.43 μg/mL, 469.58 ± 27.01 μg/mL). Notably, the protective action was more pronounced against heat-induced hemolysis (Table 5). These results surpass those reported by Haddou et al [68].
Membrane stabilizing effect of HEX-THC and CHL-CBD on hypotonicity‑induced hemolysis test and heat‑induced hemolysis test
| IC50 heat-induced test (μg/mL) | IC50 hypotonicity-induced test (μg/mL) | |
|---|---|---|
| CHL-CBD | 301.55 ± 8.43a | 469.58 ± 27.01a |
| HEX-THC | 185.94 ± 8.81b | 264.51 ± 41.18b |
| Aspirin | 100.73 ± 4.60c | — |
| Indomethacin | — | 121.63 ± 6.75c |
Data are expressed as mean value ± SD, letters within a row indicate statistically significant differences (p < 0.001), n = 3.
Exposure of RBCs to extreme conditions, such as hypotonic solutions or elevated temperatures, induces membrane disintegration, resulting in hemoglobin release and subsequent oxidation [72]. Given the structural similarity between human RBC membranes and lysosomal components, the inhibition of hemolysis under these conditions serves as a potential indicator of the extract’s anti-inflammatory mechanism. A plausible explanation for the membrane stabilization effect involves the extracts’ ability to impede the release of lytic enzymes and inflammatory mediators, thus preventing protein denaturation. Additionally, modulation of calcium influx into erythrocytes may contribute to this protective action [73]. The anti-inflammatory potential of the extracts could be attributed to the presence of bioactive compounds such as quercetin, gallic acid, p-coumaric acid, and cannabinoids, or possibly synergistic interactions between these constituents. Despite these promising findings, the precise molecular mechanisms underlying the membrane-stabilizing properties of Cannabis sativa extracts and the specific compounds responsible for this effect remain to be elucidated
Over the past few decades, numerous studies have investigated the anti-inflammatory effects of pure THC, CBD, and their combination. Research conducted both in vitro and in vivo has shown that THC can reduce inflammatory processes through multiple mechanisms. Comparable results have been observed with CBD. Majdi et al. suggested that both THC and CBD may inhibit the inflammatory response by targeting the TLR4/NF-kB signaling pathway [74]. Yekhtin et al. found that cannabis extracts had a greater inhibitory effect on cytokine secretion compared to pure cannabinoids [75]. In their study using the BV-2 mouse microglial cell line and lipopolysaccharide to induce inflammation, Kozela et al. reported that THC and CBD both diminished the production of pro-inflammatory cytokines, such as interleukin-1, interleukin-6, and interferon [76]. A recent investigation by Britch and Craft compared the effects of CBD and THC individually and in combination in male and female rats suffering from persistent inflammatory pain. Their findings indicated that THC alone significantly reduced paw thickness, while a similar effect was noted with CBD alone; however, when combined with THC, CBD appeared to lessen the effectiveness of THC [77]. Moreover, in some cases, the activity of a combination of phytomolecules was found to be superior over that of a single molecule [78,79,80].
Table 6 details the docking score of all the investigated compounds, whereas Table S1 and Figures 8–14 are the categorical descriptions, and the visual displays of various interactions involved in the hit compounds and the co-crystallized ligands of chosen target proteins, respectively. The visual display shows the three-dimensional (3D) view with the hydrogen bond surface while the two-dimensional (2D) view highlights various interacting amino acid residues. The hydrogen bonding contributes to the ligand’s stabilization near the binding site, whereas hydrophobic interactions involve non-polar amino acids, which contribute to the ligand’s binding affinity. Thus, hydrophobic interactions highlight the importance of non-polar forces in the ligand’s mode of binding (Figure 15).
Interacting amino acids, types of interactions, and binding affinity of ligands with various target proteins
| Ligands (from GC-MS and HPLC) | NF-κB | COX-1 | COX-2 | Lipoxygenase |
|---|---|---|---|---|
| 5LDE | 1EQG | 1CX2 | 3V99 | |
| Co-crystallized ligand | — | −7.6 | −9.2 | −6.0 |
| CBD | −4.6 | −6.9 | −7.7 | −7.4 |
| THC | −5.2 | −7.3 | −8.7 | −7.8 |
|
| −4.0 | −6.1 | −6.2 | −5.8 |
| Gallic acid | −3.7 | −6.6 | −6.7 | −6.3 |
| (+)-catechin hydrate | −4.8 | −7.1 | −8.4 | −8.0 |
| P-coumaric acid | −4.5 | −6.2 | −7.0 | −6.4 |
| Quercetin | −5.6 | −8.9 | −9.6 | −8.6 |
| Rosmarinic acid | −3.7 | −6.8 | −9.2 | −7.4 |
Interaction of NF-κB (5LDE) target protein with the quercetin (a) showing 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
Interaction of COX-1 (1EQG) target protein with the quercetin (b) showing 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
Interaction of COX-1 (1EQG) target protein with the co-crystallized ligand (c) showing 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
Interaction of COX-2 (1CX2) target protein with the Quercetin (d) showing 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
Interaction of COX-2 (1CX2) target protein with the co-crystallized ligand (e) showing a 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
Interaction of lipoxygenase (3V99) target protein with the quercetin (f) showing 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
Interaction of lipoxygenase (3V99) target protein with the co-crystallized ligand (g) showing a 3D amino acid interactive view along with H-Bond surface and 2D amino acid interactive view.
These findings not only deepen our understanding of the compounds’ mechanisms of action but also highlight their promising roles in various therapies. As we strive to unveil these molecular insights, boulevards for future research and therapeutic applications emerge, holding the promise of innovative approaches to enhance human health.
This study highlights the significant antioxidant and anti-inflammatory potential of CBD- and THC-enriched Cannabis sativa L. extracts, emphasizing their promising role in addressing oxidative stress and inflammation. Through comprehensive phytochemical profiling using HPLC-DAD and GC-MS, we identified key bioactive compounds, notably phenolic acids, flavonoids, and cannabinoids that contribute to these effects. The hexane and chloroform extracts, in particular, demonstrated substantial bioactivity across various in vitro assays, reinforcing their therapeutic potential. Molecular docking further elucidated the mechanisms by which these extracts may exert their anti-inflammatory effects, particularly through NF-κB inhibition and anti-lipoxygenase activity. Overall, these findings contribute valuable insights into the medicinal properties of Cannabis sativa, supporting its traditional uses and highlighting its potential for developing novel therapeutic strategies against inflammation and oxidative stress.