Determination of physicochemical properties and some biological activities of lignin, obtained from cones of the Pinaceae family using natural deep eutectic solvent

In this thesis, it was aimed to obtain lignin from Abies nordmanniana (Stev.) Spach. subsp. nordmanniana (Nordmann Fir), Cedrus libani A.Rich. (Taurus Cedar), Picea orientalis (L.) Link. (Oriental Spruce) and Pinus pinea L. (Stone Pine) cones by using natural deep eutectic solvent (NADES), which is an environmentally friendly and innovative solvent, and to reveal the chemical composition and structural properties of the obtained lignins by performing physicochemical analyzes and to determine their antioxidant, antibacterial and anticholinesterase activities. Choline chloride (ChCl) and lactic acid (LA) were used in the preparation of NADES. The treatment was carried out in a microwave reactor with ChCl:LA for 1 hour with molar ratio of 1:10, solid:liquid ratio of 1:20 and temperature of 150 °C. After the treatment, the solid (cellulose rich fraction-CRM) and liquid fractions of the samples were separated from each other. In order to make comparison with the lignin-rich fraction (LRM) samples obtained by NADES, Oriental spruce kraft lignin was produced and commercial kraft lignin was supplied. Thermogravimetric Analysis (TGA), X-Ray Diffractometry (XRD) and scanning electron microscope (SEM) analyses were carried out to determine the chemical structures of the control sample without any chemical treatment and CRM samples. Gel permeation chromatography (GPC), SEM, Fourier transform infrared (FTIR), 31P Nuclear Magnetic Resonance (31P NMR) Spectroscopy analyses were carried out for the physicochemical characterization of LRM, Oriental spruce kraft lignin and commercial kraft lignin samples. Also, the purity of LRM samples we determined with klason lignin treatment. The antioxidant activities of the obtained LRM, Oriental Spruce kraft lignin and commercial kraft lignin samples were determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. For antibacterial activities gram (-) (Klebsiella pneumoniae, Escherichia coli) and gram (+) (Enterococcus faecalis, Bacillus subtilis) strains by broth microdilution and for acetylcholinesterase (AChE) enzyme inhibition Ellman's method was used. Control samples were subjected to ethyl alcohol extraction and then klason lignin determination. Ethyl alcohol extraction and klason amounts were determined as 21.1%; 46.2% for Nordmann Fir, 24.7%; 34.7% for Taurus Cedar, 13.3%; 46.32% for Oriental Spruce and 2.9%; 36.1% for Stone Pine, respectively. CRM yields obtained after NADES treatment were found to be in the range of 42.1-63.5%. Crystallinity indices of CRM samples were higher than control samples. The purity values of LRM samples were found to be close to each other (%78.6-82.8). Lignin yields were determined as 59.2% for Nordmann Fir LRM, 89.3% for Taurus Cedar, 55.2% for Oriental Spruce and 41.4% for Stone Pine. Mw values range from 4900 g/mol to 2400 g/mol. PDI values of LRM and Oriental Spruce kraft lignin were found to be lower than commercial lignin. The highest PDI value was found in commercial kraft lignin (4.00) and the lowest in Oriental Spruce kraft lignin (2.35). According to 31P NMR analysis results, when compared with all lignin samples, the highest total phenolic OH (1.57 mmol/g) and aliphatic OH (1.92 mmol/g) and the lowest carboxylic OH (0.26 mmol/g) were detected in commercial kraft lignin. IC50 values of LRM and kraft lignins were found to be lower than synthetic antioxidant BHT, and it was concluded that their antioxidant activity was higher. It was determined that all lignin samples had both antibacterial and antiacetylcholinesterase enzyme activity at the studied concentrations. As a result, it was concluded that lignin can be obtained with high purity and yield with natural deep eutectic solvent from the cones and it has a bioactivity properties. Lignin can be use in many areas such as food, pharmacy and bioengineering. Keywords: Bioactivity, cone, lignin, microwave, natural deep eutectic solvent