Introduction
Extra virgin olive oil (EVOO) is a key component of the Mediterranean diet and is renowned for its high content of bioactive phenolic compounds that have demonstrated significant health benefits. The two prominent phenolic compounds found in EVOO are oleocanthal and oleacein. These secoiridoids are known for their anti-inflammatory, antioxidant, anticancer, and neuroprotective properties. Given their potential, considerable effort has been made to extract and purify these compounds. However, traditional extraction methods are often inefficient, environmentally harmful, and yield small quantities. To address these challenges, this study introduces a green synthesis method to produce two novel sulfur derivatives, thiocanthal and thiocanthol. These derivatives were created using a water-based sulfonation process, making them water-soluble and potentially enhancing their bioavailability and pharmacological activities.
Figure: Simple phenols (tyrosol and hydroxytyrosol) and secoiridoids (oleocanthal 1 and oleacein 2) found in EVOO.
Materials and Methods
Reagents
This study used EVOO obtained from the Coratina cultivar, a variety rich in polyphenols, to ensure high yields of oleocanthal and oleacein. Sodium metabisulfite was the primary reagent used for the sulfonation reaction, while various other chemicals and chromatography materials, such as DEAE Sepharose anion exchange resin and Affi-gel boronate affinity gel resin, were used for purification.
EVOO/Water Sulfonation Reaction
The sulfonation process is central to this study’s innovation. EVOO (200 mL) was mixed with an equal volume of 0.05% sodium metabisulfite in water and stirred magnetically for 30 min at room temperature. This process resulted in a clear separation of the two phases, with the water phase containing sulfonated products. The water-soluble products were separated from the oil phase, filtered, and monitored using UPLC-DAD-MS.
UPLC-DAD-MS Analyses
UPLC-DAD-MS analysis revealed the transformation of oleocanthal and oleacein into their sulfonated derivatives. Initially, peaks corresponding to oleocanthal and oleacein were detected at 6 and 6.5 minutes, respectively. After 30 min, these peaks diminished significantly, and two new peaks appeared at 5.1 and 5.8 minutes, corresponding to thiocanthal and thiocanthol, respectively . The mass spectra confirmed the addition of a sulfonate (-SO₃H) group to these compounds, which increased their molecular weight and confirmed the success of the sulfonation process.
Chromatographic Separation of Sulfur-Containing Products
The separation of thiocanthal and thiocanthol from the aqueous phase involved a two-step chromatographic process.
- Anion-exchange chromatography: The aqueous phase containing the sulfonated products was passed through an anion-exchange column (DEAE Sepharose), taking advantage of the ionisable sulfonate groups. The sulfonated compounds were retained on the column and eluted with phosphate-buffered saline (PBS) at pH 7.4.
- Boronate Affinity Chromatography: This step exploits the catecholic moieties of thiocanthol, which have a higher affinity for boronate-based stationary phases. The eluate from the anion-exchange column was loaded onto a boronate-affinity column. Thiocanthal, lacking the catecholic structure, eluted first with PBS at pH 7.4, whereas thiocanthol was retained and later eluted with PBS at pH 4.5.
The purity of the final products was confirmed using UPLC-DAD-MS and further purified with a hydrophobic solid-phase cartridge to remove residual salts and impurities before freeze-drying.
Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) Analyses
The structures of thiocanthal and thiocanthol were elucidated by high-resolution mass spectrometry (HR-MS) and NMR. The MS results showed intense signals at 385.0963 m/z for thiocanthal and 401.0906 m/z for thiocanthol, corresponding to their expected molecular weights after sulfonation. NMR spectra further supported these findings. The disappearance of the aldehydic hydrogen signal (9.50 ppm) in the oleocanthal confirmed the addition of the sulfonate group at position C-3 in the thiocanthal. Similar changes were observed for thiocanthol, indicating its successful sulfonation.
Figure: Phenolic secoiridoids biosynthesis pathway in Olea europaea L. (green and red arrows) and oleocanthal 1 and oleacein 2 formation during EVOO production (black arrow).
Results and Discussion
Inhibition of Cyclooxygenase (COX) Activity
The key objective of this study was to determine the anti-inflammatory potential of thiocanthal and thiocanthol compared to that of oleocanthal, oleacein, and ibuprofen. COX enzymes (COX-1 and COX-2) play crucial roles in inflammation, and their inhibition is a common target of anti-inflammatory drugs. This study found the following inhibitory effects on COX activity:
- Ibuprofen: At 50 μM, it reduced the COX activity by approximately 50%.
- Oleocanthal: Stronger inhibition, reducing COX activity by approximately 60%.
- Thiocanthal showed comparable inhibition to oleocanthal, indicating that sulfonation did not diminish its anti-inflammatory activity.
- Oleacein almost completely inhibited COX activity (approximately 96%), and thiocanthol was slightly less effective but still achieved 87% inhibition.
These results demonstrated that sulfonation did not significantly alter the bioactivity of oleocanthal and oleacein. This study thus suggests that thiocanthal and thiocanthol have potent anti-inflammatory properties and could be considered as lead compounds for novel NSAIDs.
Computational Docking Studies
To further investigate the interactions of thiocanthal and thiocanthol with COX enzymes, computational docking studies were conducted. This analysis provides insights into the potential binding affinity of these compounds to COX-1 and COX-2 binding sites. The study used the VINA software for docking and revealed the following:
- Both thiocanthal and thiocanthol displayed similar predicted binding energies to COX-2 compared to oleocanthal, oleacein, and celecoxib (a selective COX-2 inhibitor).
- Thiocanthal and thiocanthol preferentially bind to COX-2, which is important because COX-2 selective inhibitors are generally associated with fewer gastrointestinal side effects.
- Docking studies suggested that the addition of the sulfonate group might enhance the selectivity and affinity of the compounds for COX-2, making them promising candidates for safer anti-inflammatory therapies.
Conclusion
This study demonstrates a novel, green method for synthesising two sulfur-containing derivatives, oleocanthal and oleacein, thiocanthal, and thiocanthol from EVOO. By employing a sustainable aqueous sulfonation process followed by efficient chromatographic separation, researchers achieved a high-yield, environmentally friendly production of these bioactive compounds. The key findings of this study are as follows: Thiocanthal and thiocanthol possess strong anti-inflammatory properties comparable to or even exceeding those of oleocanthal, oleacein, and ibuprofen. Computational studies indicated that these derivatives have a high binding affinity for COX-2, suggesting their potential as selective COX-2 inhibitors, which could lead to fewer side effects in therapeutic applications. This green synthesis approach not only offers an innovative route for the extraction of bioactive compounds from EVOO but also opens up new possibilities for developing sustainable and effective anti-inflammatory agents. Implications and Future Research These findings will pave the way for further in vivo studies to assess the therapeutic efficacy and safety of thiocanthal and thiocanthol. Additionally, exploring their pharmacokinetic properties, bioavailability, and long-term effects will be crucial for determining their potential as new NSAIDs for clinical applications.
Read all at: Di Risola, D., Mattioli, R., Federico, R., Pascarella, G., Fontana, M., Dainese, E., Dufrusine, B., Ciogli, A., Gasparrini, F., Morea, V. and Villani, C., 2024. Green synthesis and two-step chromatographic separation of thiocanthal and thiocanthol: Two novel biologically active sulfur derivatives of oleocanthal and oleacein from extra virgin olive oil. Food Chemistry, p.141296.https://doi.org/10.1016/j.foodchem.2024.141296