Study On The Construction Of Deep Eutectic Solvent Extraction Systems And Their Applications In The Separation And Hydrolysis Mechanism Of Flavonoids From Flos Sophorae

The development of new green solvents and extraction systems is an important element in the development of green chemistry.Deep eutectic solvent（DES）is a new type of green solvent with the advantages of biodegradability,good thermal stability,low cost,and good designability.The DES-H₂O cosolvent systems constructed with diluent（H₂O）as cosolvent can reduce the viscosity and facilitate the dissolution and dispersion of solutes.The DES aqueous two-phase systems（DES-ATPS）constructed with DES as a phase-forming agent provide efficient and simple extraction systems for the separation and purification of active ingredients of Chinese herbal medicine.The flavonoids,which are abundant in Flos Sophorae,are of high medicinal value.Most of the studies on the extraction of flavonoids from plants using DES-H₂O cosolvent systems remain in the evaluation of the apparent phenomena,lacking a thorough understanding of the physicochemical properties of the systems and the interaction mechanism between the systems and flavonoids.The number of developed DES-ATPS is relatively limited,and there is still a lack of in-depth research on the phase-forming mechanism of DES-ATPS and the influence of the phase-forming ability on the partitioning behavior of flavonoids.In this paper,we designed adjustable and recyclable DES-H₂O cosolvent systems and difunctional Br?nsted acidic DES（BADES）-H₂O cosolvent catalytic systems acting as both solvent and a catalyst,and constructed novel DES/short-chain alcohols-ATPS（DES/A-ATPS）,acid-assisted DES/tert-butanol-ATPS（ADES/TBA-ATPS）and BADES-ATPS by DES and water-soluble short-chain alcohols for the separation and hydrolysis of flavonoids from Flos Sophorae.The influence of the structure and properties of the above-mentioned DES systems on the key technologies such as separation,transformation,and hydrolysis products recovery of flavonoids were investigated.The synergistic extraction and separation mechanism of DES systems on the flavonoids from Flos Sophorae was revealed at the molecular level.The main research works are as follows:（1）DES-H₂O cosolvent systems with operable temperature and adjustable hydrogen bond acidity/basicity were constructed using betaine（Bet）as hydrogen bond acceptor（HBA）,twelve natural products as hydrogen bond donor（HBD）,and green solvent（water）as diluen,and then applied to the extraction of flavonoids from Flos Sophorae.The multi-site synergistic extraction mechanism of DES-H₂O systems on flavonoids was investigated using Kamlet-Taft solvent parameters,computer simulations and infrared spectroscopy.The results showed ultrasound-assisted DES（Bet/Ure）-H₂O systems with water content of 32 wt%had an extraction yield of182.10 mg·g^-1 for rutin after 31 min of maceration at 75℃,which was 107.12 and 1.14 times higher than that of water and 70 wt%ethanol solution,respectively.The extraction process of rutin using the Bet/Ure-H₂O systems as the medium was a non-spontaneous and endothermic process,which was in accordance with a first-order kinetic model.The recovery and purity of rutin after adsorption by AB-8 macroporous resin were 64.91%and 95.37%,respectively.And the recovered DES could be recycled at least five times without significant loss of activity.The hydrogen bonding force between the Bet/Ure-H₂O systems and rutin was the main driving force for the extraction and separation of rutin from Flos Sophorae.（2）Three water-soluble short-chain alcohols,including 1-propanol（NPA）,2-propanol（IPA）,and tert-butanol（TBA）were selected as phase-forming solvents,and a novel DES/A-ATPS was constructed together with DES for the simultaneous extraction mechanism of flavonoids from Flos Sophorae.The effects of the alcohols structure,extraction temperature,and phase component concentration on the partition coefficient of rutin was investigated.The inherent relationship between the phase-forming ability of DES/A-ATPS and the interaction between the phase components and rutin was analyzed.The results showed that the extraction efficiency of rutin in DES/A-ATPS increased with the increase of phase-forming ability and tie-line length（TLL）,and the partition coefficient showed a positive correlation with its TLL value.The extraction efficiency of rutin could be increased to 92.80%using the DES/TBA-ATPS at293.15 K and atmospheric pressure,and the recovery of the systems for rutin remained above75.34%after five repetitions,with a purity of 97.46%and an average extraction amount of177.95 mg·g^-1.In the DES/A-ATPS,the extraction mechanism of rutin was dominated by the hydrogen bond interaction between the DES-riched phase and rutin,and a hydrophobic interaction between the alcohol-riched phase and the DES-riched phase was also prersent.（3）An Acid-assisted DES/TBA aqueous two-phase system（ADES/TBA-ATPS）was designed and constructed to achieve the conversion of rutin and separation of its hydrolysis products in the same system,and the hydrolysis mechanism of rutin and the competitive extraction mechanism of rutin and quercetin in this system were revealed.The results showed that the hydrolysis rate and conversion efficiency of rutin were 99.56%and 98.15%under continuous heating at 95°C and p H=2.0 for 180 min,respectively.The hydrolysis of rutin in ADES/TBA-ATPS enhanced the antioxidant activity of the extracts from Flos Sophorae with the increase of the system acidity,which could scavenge 50%of DPPH and ABTS free radicals after 39 and 27 min of hydrolysis,respectively.The hydrolysis mechanism showed that ADES/TBA-ATPS broke the glycosidic bond of rutin and converted it into quercetin,rhamnose,and glucose,and the extraction efficiency of quercetin in the TBA-riched phase was as high as99.58%.The COSMO-RS results showed that the difference in the strength of hydrogen bond interactions between rutin and quercetin with the DES-riched phase was the main reason for the difference in their competitive extraction and separation efficiency.（4）The efficient extraction of rutin,hydrolytic conversion to quercetin,and in-situ separation of hydrolysis products from Flos Sophorae were achieved using a difunctional BADES-H₂O cosolvent catalytic systems with dual functions of solvent and catalyst,and combined with BADES aqueous two-phase system（BADES-ATPS）constructed with TBA.The results showed that the hydrolysis rate,conversion efficiency of rutin,and yield of quercetin in the BADES-H₂O systems increased significantly with the increase of acid strength.The BADES（Bet/Ptsa）-H₂O systems had the highest rutin hydrolysis rate（99.84%）,conversion efficiency（100%）,and quercetin yield(3.77 mg·g^-1·min^-1)when hydrolyzed at 95℃for 60min.Compared with ADES/TBA-ATPS,the Bet/Ptsa systems presented higher conversion efficiency of rutin,yield of quercetin,and hydrolysis rate constant,as well as lower activation energy and mixing enthalpy under the same hydrolysis conditions.And its and its activity inhibition rate against DPPH,ABTS radicals and total antioxidant capacity were higher.The recovery rate of quercetin by the Bet/Ptsa-ATPS decreased by only 1.53%after five times of recovery and recycling.