Development of green extraction techniques for the determination of bioactive molecules from plant matrices

In recent years, interest in developing green extraction methods for recovering bioactive compounds from natural sources has grown significantly. This interest is driven both by the need to reduce the environmental impact of traditional chemical processes and by the urgency to valorize by-products and waste from the agri-food and industrial sectors, in line with the principles of green chemistry and the circular bioeconomy. In this context, the aim of this doctoral research was to develop and optimize analytical methods based on new “green” extraction techniques for the identification of bioactive molecules from plant matrices. Attention was focused on phenolic compounds, flavonoids, and protein pigments present in plant and microalgal sources. The research focused on developing new sustainable extraction methods, using innovative and environmentally friendly solvents such as Deep Eutectic Solvents (DESs) and their supramolecular analogues (SUPRADESs), which are considered among the most promising green solvents for applications in analytical chemistry, pharmaceuticals, and nutraceuticals. DESs are binary or ternary mixtures composed of a Hydrogen Bond Donor (HBD) and a Hydrogen Bond Acceptor (HBA), which form deep eutectic mixtures through hydrogen bonding. These solvents have unique characteristics, such as low vapor pressure, non-volatility, biodegradability, low toxicity, and adjustable polarity, making them suitable for replacing traditional organic solvents. When both components forming the DES are of natural origin, they are called Natural Deep Eutectic Solvents (NADESs). An initial research project focused on developing a “green” extraction method using DESs to extract phenolic compounds and phycocyanin (PC) from Spirulina. Optimizing the extraction parameters— including the solid/liquid ratio, temperature, and extraction time—allowed for maximizing the yield, obtaining extracts with high content of both phenolic compounds and PC through a process completely free of toxic organic solvents. Specifically, the optimized extraction procedure involved the use of a betaine-glucose NADES under the following conditions: a matrix/solvent ratio of 1:20 (w/w), a temperature of 50°C, and an extraction time of one hour. The developed procedure ensured the recovery of phenolic compounds and PC equal to 11.77 ± 1.23 mg of gallic acid equivalents (GAE) and 27.56 ± 2.46 mg per g of Spirulina powder, respectively. The PC in the NADES extract showed a slower degradation compared to the aqueous extract. Finally, the phenolic compound profile of the extract was determined by High Performance Liquid Chromatography (HPLC) coupled to mass spectrometry (MS) analysis, which allowed us to identify four phenolic compounds and two organic acids. The second research line focused on the use of SUPRADES to extract poorly soluble phenolic compounds, particularly quercetin. This flavonoid is of great interest due to its antioxidant, anti-inflammatory and anti-cancer properties. SUPRADESs are a new class of supramolecular eutectic solvents characterized by the presence of cyclodextrins (CDs). CDs are cyclic oligosaccharides known for their ability to form inclusion complexes with various guest molecules, improving their solubility, stability, and bioavailability. The experiments conducted demonstrated that SUPRADES containing β-CD can be used as extraction solvents, particularly for poorly water-soluble molecules such as quercetin. The extract was analyzed using HPLC with a Diode Array Detector (DAD). However, to evaluate the extraction capacity of these solvents, it is necessary to release the analyte from the SUPRADES. Various solvents for analyte release were tested, and the use of ethanol (EtOH) as a release solvent proved to be an ideal compromise between recovery efficiency (approximately 70%) and environmental sustainability. For both research projects, it was essential to work within a circular economy framework and in accordance with the principles of green chemistry. From a sustainability perspective, the entire proposed approach fully complies with the principles of green chemistry, as it includes: • the use of non-toxic and biodegradable solvents; • the use of low process temperatures; • the reduction of energy consumption and solvent volumes; The evidence gathered during the PhD period confirms that DES and SUPRADES represent a valid alternative to traditional organic solvents, combining analytical efficiency, selectivity, and environmental sustainability. Furthermore, the ability of DES to stabilize complex bioactive molecules, such as PC, offers the possibility to develop new formulations with extended shelf life, applicable in the pharmaceutical and nutraceutical fields. In conclusion, this research has contributed to the advancement of analytical chemistry by adhering to the principles of green chemistry and developing innovative extraction strategies that can be applied on an industrial scale. Future developments may include extending these extraction methods to new classes of bioactive molecules, further studies on the stability of SUPRADES–host molecule complexes, and the application of these SUPRADES as extraction solvents for plant matrices.