Aqueous Two-phase Systems Research Articles

Economic simulation of batch and continuous aqueous two-phase purification for viral products.

Vaccine manufacturing strategies that lower capital and production costs could improve vaccine access by reducing the cost per dose and encouraging localized manufacturing. Continuous processing is increasingly utilized to drive lower costs in biological manufacturing by requiring fewer capital and operating resources. Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technique that enables continuous processing for viral vectors. To date, no economic comparison between viral vector purifications using traditional methods and ATPS has been published. In this work, economic simulations of traditional chromatography-based virus purification were compared to ATPS-based virus purification for the same product output in both batch and continuous modes. First, the modeling strategy was validated by re-creating a viral subunit manufacturing economic simulation. Then, ATPS capital and operating costs were compared to that of a traditional chromatography purification at multiple scales. At all scales, ATPS purification required less than 10% of the capital expenditure compared to chromatography-based purification. At an 11 kg per year production scale, the ATPS production costs were 50% less than purification with chromatography. Other chromatography configurations were explored, and may provide a production cost benefit to ATPS, but the purity and recovery were not experimentally verified. Batch and continuous ATPS were similar in capital and production costs. However, manual price adjustments suggest that continuous ATPS plant-building costs could be less than half that of batch ATPS at the 11 kg per year production scale. These simulations show the significant reduction in manufacturing costs that ATPS-based purification could deliver to the vaccine industry.

A thermostable organic solvent-tolerant lipase from Brevibacillus sp.: production and integrated downstream processing using an alcohol-salt-based aqueous two-phase system.

Lipases are used for the synthesis of different compounds in the chemical, pharmaceutical, and food industries. Most of the reactions are carried out in non-aqueous media and often at elevated temperature, requiring the use of organic solvent-tolerant thermostable lipases. However, most known lipases are not stable in the presence of organic solvents and at elevated temperature. In this study, an organic solvent-tolerant thermostable lipase was obtained from Brevibacillus sp. SHI-160, a moderate thermophile isolated from a hot spring in the East African Rift Valley. The enzyme was optimally active at 65°C and retained over 90% of its activity after 1 h of incubation at 70°C. High lipase activity was measured in the pH range of 6.5 to 9.0 with an optimum pH of 8.5. The enzyme was stable in the presence of both polar and non-polar organic solvents. The stability of the enzyme in the presence of polar organic solvents allowed the development of an efficient downstream processing using an alcohol-salt-based aqueous two-phase system (ATPS). Thus, in the presence of 2% salt, over 98% of the enzyme partitioned to the alcohol phase. The ATPS-recovered enzyme was directly immobilized on a solid support through adsorption and successfully used to catalyze a transesterification reaction between paranitrophenyl palmitate and short-chain alcohols in non-aqueous media. This shows the potential of lipase SHI-160 to catalyze reactions in non-aqueous media for the synthesis of valuable compounds. The integrated approach developed for enzyme production and cheap and efficient downstream processing using ATPS could allow a significant reduction in enzyme production costs. The results also show the potential of extreme environments in the East African Rift Valley as sources of valuable microbial genetic resources for the isolation of novel lipases and other industrially important enzymes.

Extraction of collagenolytic proteases from Aspergillus heteromorphus URM 0269 in an aqueous two-phase system for application in collagen hydrolysis

Collagenolytic proteases produced by Aspergillus heteromorphus URM0269 were extracted using a PEG/sulfate aqueous two-phase system (ATPS). A 23 factorial design was performed to analyze the independent variables: PEG molar mass (MPEG), PEG concentration (CPEG), and sulfate concentration (Csulf). The extracted proteases were also evaluated for their optimum pH and stability at different pH levels (4.0 − 11.0) after 20 h of incubation. Collagen was extracted from mutton snapper (Lutjanus analis) skin using acetic acid (0.5 mol L−1). The enzyme was preferentially partitioned to the PEG-rich phase (K > 1), whose highest purification factor and recovery (PF = 6.256 and Y = 404.432%) were obtained under specific conditions: MPEG 8000 g.mol−1, CPEG 30%, Csulf 10%. The ATPS extraction provided an enzymatic activity range of pH 7.0 − 11.0, exhibiting greater stability compared to the crude extract. Approximately 80% of protease activity was maintained after 20 hours of incubation at all analyzed pH levels, except pH 11.0. Collagen extraction from L. analis skin yielded 8.056%, and both crude extract samples and ATPS-derived samples successfully hydrolyzed the extracted collagen, reaching peak hydrolysis after 36 hours of treatment. These findings demonstrate the feasibility of extracting highly purified and active proteases capable of hydrolyzing L. analis collagen.

Computational investigation on the molecular driving forces for extraction of Co2+, Ni2+, and Fe3+ in poly(ethylene oxide)/thiocyanate salt aqueous two-phase systems

Aqueous two-phase systems are environmentally friendlier alternatives for hydrometallurgical processing and purification of metals. However, the viability of application of those systems in such processes is still under investigation, in which the comprehension of the fundamental aspects that govern the metal extraction is essential and can be further constructed using computational approaches. This research paper presents a comprehensive computational study on the fundamental nature of metal extraction in polymer-salt aqueous two-phase systems (ATPS), comparing the results of modeling and simulation work to the theoretical and experimental data available in the literature. Specifically, the mechanisms underlying the extraction of Co2+, Ni2+, and Fe3+, in ATPS formed by poly(ethylene oxide) (PEO) and thiocyanate salt are investigated, using quantum chemical calculations and molecular dynamics simulations. The formation of pseudo-polycations (PPCs) and thiocyanate metalates (complexes formed by a metal cation bonded to one or more anions) and the interaction of PEO fragments, thiocyanate, and aquo ligands with metal cations are explored. The binding affinity of these species is evaluated from various perspectives, including structural, electronic, and thermodynamic considerations. The solvation behavior of the species in solvents with different polarities is also examined. The findings elucidate the mechanisms governing metal cations partitioning behavior in PEO/thiocyanate ATPS, confirming the mechanisms proposed previously in the literature, involving charge transfer between metal-thiocyanate complexes and polyethylene oxide ether groups as well as interactions between metalate and PPCs. This study represents the first-ever computational investigation into the molecular driving forces behind metal extraction in polymer-salt ATPS, providing valuable insights for optimizing extraction processes and paving the way for future research in this field.

Synthetic Membraneless Droplets for Synaptic-Like Clustering of Lipid Vesicles.

In eukaryotic cells, the membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) are found to interact intimately with membranous organelles (MOs). One major mode is the clustering of MOs by MLOs, such as the formation of clusters of synaptic vesicles at nerve terminals mediated by the synapsin-rich MLOs. Aqueous droplets, including complex coacervates and aqueous two-phase systems, have been plausible MLO-mimics to emulate or elucidate biological processes. However, neither of them can cluster lipid vesicles (LVs) like MLOs. In this work, we develop a synthetic droplet assembled from a combination of two different interactions underlying the formation of these two droplets, namely, associative and segregative interactions, which we call segregative-associative (SA) droplets. The SA droplets cluster and disperse LVs recapitulating the key functional features of synapsin condensates, which can be attributed to the weak electrostatic interaction environment provided by SA droplets. This work suggests LLPS with combined segregative and associative interactions as a possible route for synaptic clustering of lipid vesicles and highlights SA droplets as plausible MLO-mimics and models for studying and mimicking related cellular dynamics.

Recovery of superbase ionic liquid using aqueous two-phase systems

Aqueous two-phase systems (ATPS) have been widely used for the purification and separation of biomolecules. However, recent studies have shown that ATPS can also be effective in the purification and recovery of other compounds, such as ionic liquids (IL). Here, we investigated the liquid-liquid phase equilibria of ATPS consisting of 5-methyl-1,5,7-triaza-bicyclo[4.3.0]non-6-enium acetate ([mTBNH][OAc]) and potassium salts (K2CO3 and K3PO4) at 293.2 K and 323.2 K. Our results indicate that both systems exhibited a high IL partition coefficient (k), exceeding 9.5, and achieved a 99.5% recovery of ionic liquid. Moreover, we found that the presence of potassium salt in the ionic liquid-rich phase had a negative impact on the IL cellulose dissolution capability. Therefore, we investigated the removal of potassium salt from the IL and successfully achieved it by using a nanoporous sodium zirconium cyclosilicate (ZS-9 or AV-13). Overall, our study suggests that ATPS can be a promising technique for the purification and recovery of ionic liquids, with potential applications in various industries.

Equilibrium phase behavior of ternary systems formed by polyethylene glycol + choline salts + water

The economic and environmental potentialities of the application of choline salts in aqueous two-phase systems (ATPS), as well as the incipiency of works of these ionic liquids (ILs) combined with polyethylene glycol (PEG), are the reasons for the characterization of new “green” ATPS formed by PEG (400 and 1500 g.mol−1) combined with choline-based ILs (choline chloride, choline bitartrate, and choline dihydrogen citrate). Choline chloride was preferentially concentrated in the top phase, while the lower phase was rich in PEG. The opposite behavior was observed for bitartrate and dihydrogen citrate. ATPS formed by PEG1500 exhibits a significant increase in the biphasic area as the temperature increases; while for systems formed by PEG400, this same trend is slight, demonstrating that phase segregation has an endothermic character. The salting-out effect and the hydrophobicity of the polymer explain the increase of the biphasic area with increasing the charge density of the anion and increasing polymer molar mass, respectively. The data were fitted with good correlation using the Merchuk equation for the binodal curves and Guan equation for the tie-lines.

Aqueous two-phase systems coupled with chemometrics-enhanced HPLC-DAD for simultaneous extraction and determination of flavonoids in honey.

In this study, an accurate, rapid, green, and environment friendly method for the extraction and quantitative analysis of flavonoids in honey was established by using the aqueous two-phase extraction combined with the chemometrics-assisted HPLC-DAD. The first purpose of this study was to extract seven flavonoids in five different types of honey using alcohol/salt aqueous two-phase system (ATPS). The system with 2.82mL sodium citrate (30%), 1.58mL water, and 3.10mL isopropanol, showed the highest flavonoids extraction yields in the top phase (87.66-101.50%). Additionally, the three-way array of honey samples based on HPLC-DAD was decomposed mathematically by the alternating trilinear decomposition (ATLD) algorithm to obtain reasonable chromatograms, spectra, and concentration profiles for each analyte. Compared with the traditional solid-phase extraction method, the ATPS-ATLD-based method showed satisfactory spiked recoveries, lower limit of detection, and higher sensitivity, further verifying its accuracy and stability.

About phase separation of aqueous polymeric two-phase dextran-polyvinylpyrrolidone systems

About phase separation of aqueous polymeric two-phase dextran-polyvinylpyrrolidone systems

Microfluidic Aqueous Two-Phase Focusing of Chemical Species for In Situ Subcellular Stimulation.

Confinement of chemical species in a controllable micrometer-level (several to a dozen micrometers) space in an aqueous environment is essential for precisely manipulating chemical events in subcellular regions. However, rapid diffusion and hard-to-control micrometer-level fluids make it a tough challenge. Here, a versatile open microfluidic method based on an aqueous two-phase system (ATPS) is developed to restrict species inside an open space with micron-level width. Unequal standard chemical potentials of the chemical species in two phases and space-time correspondence in the microfluidic system prevent outward diffusion across the phase interface, retaining the target species inside its preferred phase flow and creating a sharp boundary with a dramatic concentration change. Then, the chemical flow (the preferred phase with target chemical species) is precisely manipulated by a microfluidic probe, which can be compressed to a micron-level width and aimed at an arbitrary position of the sample. As a demonstration of the feasibility and versatility of the strategy, chemical flow is successfully applied to subcellular regions of various kinds of living single cells. Subcellular regions are successfully labeled (cytomembrane and mitochondria) and damaged. Healing-regeneration behaviors of living single cells are triggered by subcellular damage and analyzed. The method is relatively general regarding the species of chemicals and biosamples, which could promote deeper cell research.

Evaluation of ethanol-based aqueous two-phase systems performance for application in centrifugal partition chromatography (CPC)

Aqueous two-phase systems (ATPS) have recently been proposed for Centrifugal Partition Chromatography (CPC). This type of biphasic system has several advantages over conventional aqueous-organic liquid–liquid systems, namely, high biocompatibility owing to its large water content. The most commonly used ATPS are polymer-based systems. However, owing to their poor phase separation hydrodynamics compared to aqueous-organic systems, the use of short-length water-miscible alcohols has been evaluated as an alternative to polymers. In this study, an ATPS composed of ethanol and dipotassium hydrogen phosphate (K2HPO4) is proposed to overcome the common hydrodynamic drawbacks of polymer-based ATPS. The Ethanol/K2HPO4 system exhibited a very short settling time (ts = 30.85 ± 0.80 s), high interfacial tension (IFT = 6.38 mN/m), significant density differences between the phases (Δρ = 353.40 kg/m3), and low viscosity of the coexisting phases at 25 °C (maximum µ = 3.79 ± 0.11 mPa.s), making it an ideal choice for implementation in CPC apparatus. A comprehensive study of this biphasic system was conducted using CPC equipment by carefully adjusting the operating parameters, specifically, the mobile phase flow rate (F), rotational speed (ω), and mode of operation, that is, ascending (upper phase is mobile) or descending (lower phase is mobile). In this particular CPC equipment, the use of polymers as phase-forming compounds in ATPS resulted in higher CPC pump pressures compared to ethanol or phosphate salt aqueous solutions, thus confirming the superior performance of the ethanol/salt ATPS. The results obtained using this ATPS demonstrated that high F and ω values lead to lower stationary phase retention (SF) and higher backpressures, respectively. SF remained relatively constant and above 50% for F ≤ 6 mL/min but significantly decreased for F > 6 mL/min. This trend confirms that hydrostatically stable conditions can be achieved by employing the Ethanol/K2HPO4 system in the CPC mode by controlling F (i.e., the hydrodynamics of the mobile phase). The partition of the model solutes gallic acid and L-tyrosine was also evaluated using the ethanol/K2HPO4 system. The critical operating CPC parameters were compared, confirming the significant potential of the ethanol/salt ATPS for successful commercial application of this chromatographic technique in the separation of small value-added biomolecules.

Thermodynamic modelling of the partitioning of lysozyme in aqueous two-phase systems composed of polyethylene glycol and salt at different temperatures

Knowledge about the interactions during the protein transfer is necessary to optimize the application of aqueous two-phase systems (ATPS) in purification processes. Theoretical knowledge combined with modelling and simulation can help to select the systems with the best partition values. Therefore, this study aimed to determine new interaction parameters through the contribution groups of lysozyme, using UNIFAC (Universal Functional Activity Coefficient) modelling. First, the partition of lysozyme was carried out, and the polymer molar mass (4000 or 6000), and the effects of the type of salt (Na2SO4 or Na3C6H5O7), the overall composition of the system (3 mooring lines), and temperature T = (293.15, 298.15, 303.15 and 308.15) K were evaluated. The results showed that lysozyme had a preference for the bottom phase of the ATPS system, the increase in the overall composition led to a tendency of increasing the partition coefficient (Kp), and the systems made up of higher molar mass polymers exhibited a bottom Kp value. The results of the thermodynamic parameters showed that the transfer of lysozyme in the phases was governed by entropy contribution. New interaction parameters of the contribution groups of lysozyme in the system phases were estimated. The UNIFAC model showed low deviation values. Therefore, the present results allow estimating new partitioning data for proteins presenting the same contribution groups, which can reduce the number of experiments and optimize the partitioning process.

Extraction of functional natural products employing microwave-assisted aqueous two-phase system: application to anthocyanins extraction from mulberry fruits

Aqueous two-phase extraction (ATPE) has been extensively utilized for the extraction and separation of tiny-molecule substances as a new system (system with short-chain ethanol and inorganic salts). In this study, an innovative method of extracting anthocyanins from mulberry was developed, employing microwave-assisted extraction with ethanol/ammonium sulfate as a biphasic extractant. Response surface methodology (RSM) was utilized to optimize anthocyanin extraction conditions: 39% ethanol (w/w), 13% ammonium sulfate (w/w), and liquid-to-solid ratio of 45:1, microwave duration 3 min, microwave temperature 32 °C, and microwave power 480 Watt (W). High-performance liquid chromatography (HPLC) analysis demonstrated no significant differences in the structure of mulberry anthocyanins before and after MAATPE treatment, furthermore. The extraction behavior of MAATPE was due to hydrogen bonding, according to Fourier transform infrared spectroscopy (FT-IR). Scanning electron microscopy analysis found that MAATPE damaged the cell structure via a microwave enhancement effect, which was more favorable to anthocyanin dissolution than standard extraction methods. The DPPH free radical scavenging rate of mulberry extracts at 0.5 mg/mL was higher than that of vitamin C (96.4 ± 0.76%), and the ABTS free radical scavenging rate (82.52 ± 2.13%) was close to that of vitamin C, indicating that MAATPE-derived mulberry extracts have good antioxidant activity.

Cobalt and nickel separation in aqueous two-phase systems with polyethylene glycol 20,000 and sodium electrolytes

The separation of Co(II) and Ni(II) in aqueous two-phase systems (ATPSs) formed by the polymer polyethylene glycol 20,000 and several sodium electrolytes, coupled with the organic extractant 1-nitroso-2-naphthol was studied. First, the liquid-liquid equilibrium experiments were carried out to describe the phase diagrams of the ATPS. Then, the metal separation efficiency was evaluated considering several factors. It was observed that pH had a great influence on the selective metal chelation. When lowering the pH to 2 and 1 in the system with sodium sulfate, it was possible to suppress the Ni extraction under 5%. These results showed that the differences in the metal chelate stabilities could displace the equilibrium reaction with Ni(II) for selective extraction. In addition, experimental results showed that the interactions between metal cations and the salt anions also determined the separation efficiency. For the system with sodium sulfate (Na2SO4), Ni(II) extraction was suppressed at pH 2, but when sodium citrate (Na3C6H5O7) was used, the suppression occurred at pH lower than 0.5, while sodium tartrate (Na2C4H4O6) and sodium hydrogen phosphate (Na2HPO4) were not suitable for selective extraction. High separation factors of 11,080, 1136, and 3300 were obtained for the aqueous systems with Na2SO4 at pH 2, and pH 1, and Na3C6H5O7 at pH 0.5, respectively. Finally, the extraction at higher metal ion concentrations was tested. When the concentration of 1N2N was 1000 mg/kg, the maximum amount of extracted Co reached about 85 mg/kg within 15 min, which corresponds to complete extraction considering a metal:1N2N ratio of 1:4.

Data-Driven Imputation of Miscibility of Aqueous Solutions via Graph-Regularized Logistic Matrix Factorization.

Aqueous, two-phase systems (ATPSs) may form upon mixing two solutions of independently water-soluble compounds. Many separation, purification, and extraction processes rely on ATPSs. Predicting the miscibility of solutions can accelerate and reduce the cost of the discovery of new ATPSs for these applications. Whereas previous machine learning approaches to ATPS prediction used physicochemical properties of each solute as a descriptor, in this work, we show how to impute missing miscibility outcomes directly from an incomplete collection of pairwise miscibility experiments. We use graph-regularized logistic matrix factorization (GR-LMF) to learn a latent vector of each solution from (i) the observed entries in the pairwise miscibility matrix and (ii) a graph where each node is a solution and edges are relationships indicating the general category of the solute (i.e., polymer, surfactant, salt, protein). For an experimental data set of the pairwise miscibility of 68 solutions from Peacock et al. [ACS Appl. Mater. Interfaces 2021, 13, 11449-11460], we find that GR-LMF more accurately predicts missing (im)miscibility outcomes of pairs of solutions than ordinary logistic matrix factorization and random forest classifiers that use physicochemical features of the solutes. GR-LMF obviates the need for features of the solutions and solutions to impute missing miscibility outcomes, but it cannot predict the miscibility of a new solution without some observations of its miscibility with other solutions in the training data set.

Recovery and partial purification of fibrinolytic protease from Pleurotus ostreatus and P. eryngii and cytotoxic and antioxidant activity of their extracts

Mushrooms are a source of primary and secondary metabolites. Little is known about the most suitable conditions for production of mushrooms by submerged fermentation. This article reports antioxidant and cytotoxic assays, in addition to quantitatively evaluating the content of proteases with fibrinolytic action in the crude extracts of two species of edible mushrooms produced in different formulations, as well as evaluating the recovery of these enzymes by aqueous two-phase systems (ATPS). The mushrooms Pleurotus ostreatus and Pleurotus eryngii, at concentration of 100 µg/mL, displayed inhibition of DPPH and ABTS radicals below 50%. In the cytotoxicity test, the cells human fibroblast cell lines (MRC-5) showed cell viability greater than 80%. Concerning fibrinolytic activity, P. eryngii presented 226.47 ± 7.26 U/mL, therefore being more efficient than P. ostreatus (71.5 ± 0.56 U/mL). In the recovery of the P. eryngii extract by ATPS, the fibrinolytic protease was partitioned in the salt phase (30.25 U/mL). The molecular mass of the proteases was between 75 and 100 kDa. These results prove the low cytotoxicity of the extracts produced and that fermentation in supplemented malt broth favored the excretion of fibrinolytic proteases compared to the other evaluated media.

Aqueous two-phase extraction and characterization of thermotolerant alkaliphilic Cladophora hutchinsiae xylanase: biochemical properties and potential applications in fruit juice clarification and fish feed supplementation

Xylanase finds extensive applications in diverse biotechnological fields such as biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. Here, polyethylene glycol (PEG)-phosphate aqueous two-phase system (ATPS) was applied for the purification of an alkaline active and thermotolerant xylanase from a marine source, Cladophora hutchinsiae (C. hutchinsiae). In the purification process, the effects of some experimental factors such as PEG concentration and PEG molar mass, potassium phosphate(K2HP04) concentration, and pH on xylanase distribution were systematically investigated. Relative enzymatic activity and purification factor obtained were 93.21% and 7.18, respectively. A single protein band of 28 kDa was observed on SDS-PAGE. The optimum temperature and pH of xylanase with beechwood xylan were 30 °C and 9.0, respectively. The Lineweaver-Burk graph was utilized to determine the K m (4.5 ± 0.8 mg/mL), V max (0.04 ± 0.01 U) and k cat (0.001 s−1) values of the enzyme. It was observed that the purified xylanase maintained 70% of its activity at 4 °C and was found stable at pH 4.0 by retaining almost all of its activity. Enzymatic activity was slightly enhanced with Na+, K+, Ca2+ and acetone. The highest increase in the reducing sugar amount was 53.6 ± 3.8, for orange juice at 50 U/mL enzyme concentration.

Curcuminoid extraction from rhizomes of Curcuma longa via aqueous two phases system

The extraction of curcuminoids [curcumin (C21H20O6, CUR), demethoxycurcumin (C20H18O5, DMC), and bisdemethoxycurcumin (C19H16O4, BDMC)] from Curcuma longa rhizomes was investigated using an aqueous two-phase system (ATPS) with ethanol/salt. The optimized conditions for the extraction process were determined by a central composite design to be an ethanol concentration of 13.6%, an ammonium sulfate concentration of 33.2%, and a temperature of 66.8 °C. Under these conditions, the highest yield of 97.9% was achieved, accompanied by a notable antioxidant activity of 84.38%. The experimental data exhibited good agreement with a second-order kinetic model, as confirmed by the evaluation of the coefficient of determination (R 2), normalized standard deviation, and average relative error.

Ionic liquid-assisted aqueous two-phase system for the enrichment of multiple monosaccharides from jujube

In this study, a simple, high-efficiency and eco-friendly enrichment method using an ionic liquid (IL)-assisted aqueous two-phase system was proposed for the enrichment of multiple monosaccharides. The parameters affecting the enrichment effect were the type of IL, the mass of the IL, the amount of salt, the vortex time, and the pH value of the solution. All of these parameters were optimized using single-factor tests or multifactor experiments. The optimal conditions were as follows: [C4min]Br was 313.49 mg, the mass of K2HPO4 was 4.6 g, the vortex time was 3.18 min, and the pH value was 9. In addition, the established method was verified to have good linearity (r2 ≥ 0.9991) and high sensitivity, with limits of detection ranging from 6.1 to 34.09 ng/mL and limits of quantification ranging from 20.57 ng/mL to 113.63 ng/mL. Spiked samples had recoveries ranging from 85.68 % to 104.83 %. The present work demonstrated that this established method can be successfully used for the extraction and enrichment of various monosaccharides from jujube.

Study on Extraction and Purification of Acanthopanax senticosus Polyphenols by an Ionic Liquid-Assisted Aqueous Two-Phase System.

This study aimed to extract and purify polyphenols from Acanthopanax senticosus. A new green method was developed, in which ionic liquids (ILs) were used as aqueous two-phase (ATP) adjuvants to extract the polyphenols from A. senticosus. An ionic liquid-assisted aqueous two-phase system (IL-ATPS) was established. The purification of the polyphenols from the extraction fluid by AB-8 macroporous resin was conducted, and the kinetic mechanisms were studied. The reuse of ionic liquids was executed. The results showed that an [OMIM]Br-assisted ethanol/NaH2PO4 system (IL-ATPS) was the best extraction solvent. In this study, the following optimal extraction conditions were determined: 32 wt.% ethanol, 25 wt.% NaH2PO4, 9 wt.% additional ionic liquid, a solid-liquid ratio of 1:40 g/mL, an extraction temperature of 50 °C, a pH of 4.0, an extraction time of 50 min, and an extraction rate of the polyphenols at 15.90 mg/g. The optimum adsorption parameters of the macroporous resin AB-8 were as follows: a flow rate of 3.5 BV·h-1, a sample volume of 40 mL, an elution flow rate of 3.5 BV·h-1, an eluent volume of 80 mL, and an eluant that was constituted by an 85% volume fraction of ethanol. The decolorization effect of 4% activated carbon was better than the other amounts; in addition, a decolorization rate of 76.81% and an ionic liquid recovery rate of 81.12% were found to be the most optimal. Compared with the traditional extraction methods, IL-ATPS has the advantages of requiring simple operation, saving time, and high efficiency. In addition, it can be used for the extraction of the polyphenolic compounds.