Efficient Extraction And Separation Processes Of Polyphenolic Compounds From Lingonberry Fruit Pomace

Lingonberry（Vaccinium vitis-idea Linn.）is a terrestrial wild shrub belonging to the genus Vaccinium spp.in the family Ericaceae.The lingonberry is rich in nutrients and has many beneficial biological activities.In this thesis,six polyphenolic components of lingonberry pomace were extracted from the lingonberry pomace by forming a CO₂-responsive in situ deep eutectic solvent and separated by high speed counter-current chromatography（HSCCC）coupled with macroporous adsorbent resin.This study provides technical support for the high value processing and utilization of lingonberry.The research content and results are as follows:High performance liquid chromatographic（HPLC）method was developed for the determination of five model compounds（catechin,quercetin,gallic acid,oleanolic acid and citric acid）in the crude extract of lingonberry fruits.The methodological validation showed that the established HPLC method had good linearity with R² greater than 0.999 in the range of 5-500μg/m L,and the detection limits of the five model compounds were in the range of 0.35-0.51μg/m L and the quantification limits were in the range of 1.15-1.74μg/m L with high sensitivity.The intra-day and inter-day precision was good and reproducible.In addition,the comparative analysis of the juice and pomace content of lingonberry provided favorable data to support the resource utilization of lingonberry.In order to verify the formation of an in situ deep eutectic solvent with solubilizing effect with plant secondary metabolites based on solvent extraction,the intermolecular interactions between solvent ethanolamine and plant secondary metabolites during the extraction process were investigated,and typical components of lingonberry pomace（catechin,quercetin,gallic acid,citric acid and oleanolic acid）were selected as model compounds for synthesis with ethanolamine.The formation of hydrogen bonds was verified by Fourier infrared spectroscopy;the glass transition temperatures of the prepared mixture models were measured by differential scanning calorimetry to be lower than the melting points of the individual compounds;the non-covalent interactions between the single model compounds and ethanolamine molecules were calculated by density functional theory,and the electron density at the bond saddle point and the Laplace function of electron density were found to be within the range of hydrogen bonds.The results showed that the electron density and the Laplace function of electron density at the bond saddle point were all within the range of hydrogen bonding,which favorably confirmed that the five mixture models were deep eutectic solvents and verified the hypothesis of in situ deep eutectic solvent formation.Moreover,the desorption test revealed that the model system consisting of five model compounds with a wide variety of polyphenols,triterpenoids,and organic acids changed to a system with polyphenols as the main component and a significantly lower content of other components,which confirmed the targeting of polyphenols by the extraction process.The ultrasound-assisted extraction time,liquid-solid ratio,system water content and ultrasound power were optimized by single-factor test,and then the process parameters for the extraction of polyphenols in lingonberry pomace were jointly investigated and optimized by Box-Behnken Design interaction,and the best extraction process parameters were determined by fitting the quadratic regression equation:the ultrasound extraction time is 16 min,the liquid-solid ratio is 18 m L/g,the system water content is 12 wt%,and the ultrasound power was 400 W.The extraction yields of total phenols,catechin,quercetin and gallic acid under these conditions were 3.264 mg/g,0.479 mg/g,0.024 mg/g and 0.062 mg/g,respectively.The extraction efficiency of the CO₂ responsive in situ deep eutectic solvent was verified against21 natural deep eutectic solvents successfully synthesized,confirming the high efficiency of the method,and the recovery test confirmed that the ethanolamine recovered after a simple step could guarantee at least five cycles until the fifth cycle was 87%of the original extraction yield.The macroporous adsorbent resin was used to enrich the polyphenols in the crude extract of lingonberry,and the optimal enrichment process was explored:the resin type was AB-8 type macroporous adsorbent resin,the loading volume was 60 m L,the loading flow rate was 1 BV（Resin Bed Volume,BV）/h,the concentration of debinding eluent solution:40%ethanol solution,the volume of debinding eluent solution:6 BV,the concentration of eluent solution:The enrichment of the polyphenols in the crude extracts of lingonberry was carried out by using the optimized process parameters of adsorption and desorption on the macroporous adsorbent resin,and the total phenolic content after enrichment was 57.85%,which was twice of that before enrichment（27.1%）.High-speed counter-current chromatography was applied to prepare and separate six polyphenolic compounds in lingonberry pomace with the following process parameters:First separation solvent system:n-butanol/methyl tert-butyl ether/acetonitrile/water/TFA（2:2:1:6:0.01,v/v）,second separation solvent system:n-butanol/ethyl acetate/water（1:9:10,v/v）,thermostatic circulator:25℃,loading volume:150 mg,mobile phase flow rate:1.5 m L/min,mainframe speed:850 r/min..The above optimized process conditions were used for the separation and preparation,and after the analysis of the retention time and mass spectrometry data of the monomer compounds by high performance liquid chromatography and mass spectrometry,it was concluded that after two high-speed counter-current separation and preparation,six high-purity polyphenolic monomers could be obtained,which were cyanidin-3-O-galactoside,cyanidin-3-O-glucoside,cyanidin-3-O-arabinoside,catechin,quercetin-3-O-galactoside,quercetin-3-O-rhamnoside.The purity was 89.6%,91.4%,86.3%,83.5%,86.2%and 91.6%by HPLC peak area normalization method,respectively.In summary,a novel extraction system,CO₂ responsive in situ deep eutectic solvent,was developed in this paper for the extraction of polyphenolic compounds from rhodopsin,and the formation of in situ deep eutectic solvent was verified by a combination of instrumentation and quantum chemical calculations.The specific desorption of polyphenolic compounds can be accomplished by passing CO₂.The crude extract of lingonberry enriched with macroporous resin was prepared by high-speed counter-current chromatography,and the separation of six polyphenolic monomers was completed within five hours.This study developed a new,"green"and efficient process for the extraction,enrichment and separation of polyphenolic compounds,which provides a new idea for the utilization of lingonberry resources and the preparation of polyphenolic compounds.