Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

BACKGROUND PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating Nterminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. RESULTS: Selected linear 5 kDa and 20 kDa mPEG ­ potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the diPEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods.

Optimization of Ultrasonic-assisted Ethanol- （NH4）2SO4 Aqueous Two-phase Extraction of Citrusinol from Desmo- dium caudatum by Box-Behnken Design-response Surface Methodology / 中国药房

OBJECTIVE：To optimize ultrasonic-assisted ethanol-（NH4）2SO4 aqueous two-phase extraction technology of citru- sinol from Desmodium caudatum . METHODS ：Using the content of citrusinol as indexes ，with ethanol volume fraction ， solid-liquid ratio ，（NH4）2SO4 addition amount ，ultrasonic time and ultrasonic temperature as factors ，based on the single factor tests，Box-Behnken design-response surface methodology was used to optimize the extraction technology of citrusinol. RESULTS ： The optimized extraction technology of citrusinol included that ethanol volume fraction was 95.35％，the solid-liquid ratio was 1∶50.35 （g/mL），（NH4）2SO4 addition amount was 4.49 g，ultrasonic time was 48.7 min，ultrasonic temperature was 57.6 ℃. In 3 times of validation tests ，the extraction rates of citrusinol were 0.637 8，0.638 4，0.625 4 mg/g，respectively，which was close to predicted value（0.630 5 mg/g）. CONCLUSIONS ：The optimized ultrasonic-assisted ethanol- （NH4）2SO4 aqueous two-phase extraction technology is stable and feasible ，and can be used for the extraction of citrusinol from D. caudatum .

Optimization of two-phase aqueous extraction for Astragali Radix by central composite design-response surface method / 中成药

AIM To optimize the two-phase (ethanol-dipotassium phosphate) aqueous extraction for Astragali Radix by central composite design-response surface method.METHODS With ethanol concentration,dipotassium hydrogen phosphate solution concentration and Astragali Radix feed ratio as influencing factors,together with extraction rates of total flavonoids and total saponins as evaluation indices,the technology optimization was accomplished by central composite design-response surface method.RESULTS The optimal conditions were determined to be 27.97％ for ethanol concentration,22.03％ for dipotassium hydrogen phosphate solution concentration,and 1 ∶ 58.85 for Astragali Radix feed ratio.The extraction rate of total flavonoids reached 74.13％ (concentrated in the upper layer),while that of total saponins reached 81.34％ (concentrated in the lower layer).CONCLUSION With good predictability,this simple and accurate method can be used for the extraction of total flavonoids and total saponins in Astragali Radix.

Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of β-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD+-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.

Advances in application of aqueous two-phase system in separation and purification of active constituents from Chinese materia medica / 中草药

Aqueous two-phase system (ATPS) is a new liquid-liquid fractionation technique that has been extensively applied in the separation and purification of natural products including proteins, genetic material, bionanoparticles, cells, and secondary metabolites. Advantages of the technique include scale-up potential, continuous operation, ease of process integration, low toxicity, etc. This paper reviewed the applications in separation and purification of the active constituents from Chinese materia medica (CMM) in recent five years, so as to promote the development of separation of the active constituents in CMM.

Separation and enrichment of geniposidic acid from leaves of Eucommia ulmoides by aqueous two-phase flotation / 中草药

Objective: Ethanol/NaH2PO4 aqueous two-phase gas solvent sublation coupled with response surface methodology was developed for the separation/enrichment and analysis of geniposidic acid from the leaves of Eucommia ulmoides. Methods: For the aqueous two-phase flotation project, the flotation effects of different solvent, salts, flotation rate, flotation time, amount of crude extract and so on were deeply investigated; Furthermore, a Box-Behnken central composite test design was studied to determine the best flotation process conditions of GPA, and three factors including the mass fraction of NaH2PO4, the mass fraction of ethanol, and the flotation rate were selected based on the single factor test in the design. A 100 times scale-up experiment also was tested. Results: The optimal conditions of the flotation were as follows: the mass fraction of NaH2PO4 was 25%, the mass fraction of ethanol was 20%, the amount of crude extract was 5 g, the flotation rate was 30 mL/min, and the flotation time was 20 min. Under the optimal condition, the flotation efficiency was 97.88%, and the enrichment factor was 27.34; The 100 times scale-up results showed that the flotation efficiency of GPA was 95.60%, and the RSD value was 0.77%. Conclusion: The method with ethanol/NaH2PO4 aqueous two-phase gas solvent sublation is suitable for the separation and enrichment of the active ingredients from E. ulmoides because of its high distribution coefficient and large enrichment factor.

Aqueous Two-phase Systems with Ultrasonic Extraction Used for Extracting Phenolic Compounds from Inonotus obliquus / 中草药·英文版

Objective: To optimize the extracting technology of assessing the maximum yield of phenolic compounds (PC) from Inonotus obliquus by single factor experiments and orthogonal array design methods through aqueous two-phase systems combined with ultrasonic extraction. Methods: The range of the independent variables, namely levels of acetone and ammonium sulfate, and ultrasonic time were identified by a first set of single factor experiments. The actual values of the independent variables coded at four levels and three factors were selected based on the results of the single factor experiments. Subsequently, the levels of acetone and ammonium sulfate, and ultrasonic time were optimized using the orthogonal array method. Results: The optimum conditions for the extraction of PC were found to use 7.0 mL acetone, 5.5 mg ammonium sulfate, with ultrasonic time for 5 min. Under these optimized conditions, the experimental maximum yield of PC was 37.8 mg/g, much higher than that of the traditional ultrasonic extraction (UE, 29.0 mg/g). And the PC obtained by this method had stronger anti-oxidative activities than those by traditional UE method. Conclusion: These results indicate the suitability of the models developed and the success in optimizing the extraction conditions. This is an economical and efficient method for extracting polyphenols from I. obliquus. © 2013 Tianjin Press of Chinese Herbal Medicines.

Extração de bromelina dos resíduos de abacaxi ( Ananas comosus ) por sistemas de duas fases aquosas e sua aplicação em hidrogel polimérico / Extraction of bromelain from pineapple waste ( Ananas comosus ) by aqueous two-phase systems and its application in polymeric hydrogels

Bromelina é um nome coletivo para enzimas proteolíticas encontradas no talo, fruto e folhas do abacaxi (Ananas comosus Merr). A bromelina possui propriedades anti-inflamatórias, de debridamento, entre outras. Para a produção da bromelina deve-se, preferencialmente, usar resíduos do abacaxi, visto que os produtos do fruto têm aplicação comercial. Este trabalho teve como objetivo a extração de bromelina a partir de cascas de abacaxi através de sistema de duas fases aquosas (SDFA), e sua aplicação em hidrogel polimérico. Foram realizados estudos de estabilidade da bromelina comercial, em que se observou maior estabilidade em pH 5,0 com menor perda da atividade relativa em todas as temperaturas estudadas (20, 30, 40 e 50°C). O estudo da extração da bromelina em SDFA formado por polietileno glicol (PEG) e ácido poliacrílico (PAA) (com auxílio da análise de variância de parâmetros como rendimento, fator de purificação e coeficiente de partição) proporcionou rendimento de 335% e fator de purificação de 25,8. Os hidrogéis poliméricos à base de PEG estudados apresentaram-se flexíveis, com pouca elasticidade e taxa de absorção superior a 1000%. Hidrogel carreado de bromelina pelo método de turgescência proporcionou a maior liberação da enzima, assim como a maior atividade (80% da bromelina liberada em 24 h e 278 ± 89 U/mL)

Bromelain is a collective name for the proteolytic enzymes found in the stem, fruit and leaves of pineapple (Ananas comosus Merr.). Bromelain possesses anti-inflammatory properties, debridement, among others. For bromelain production one should preferably use the waste materials, whereas pineapple fruit products have commercial application. This study aimed to extract bromelain from pineapple peels using aqueous two-phase system (ATPS), and its application in polymeric hydrogels. Stability studies of commercial bromelain were performed, which found greater stability at pH 5.0 with minor loss of relative activity at all temperatures studied. The study of bromelain extraction in ATPS composed by polyethylene glycol (PEG) and poly acrylic acid (PAA) (with assistance of variance analysis of parameters such as yield, purification factor and partition coefficient) showed yield 335% and purification factor of 25.8. The PEG-based hydrogels studied presented flexibility, low elasticity and swelling ratio higher than 1000%. Hydrogel containing bromelain, loading by embedding (solvent sorption) method, yielded the highest enzyme release, as well as the highest activity (80% bromelain released over 24 h and 278 ± 89 U / mL)

Extraction of lipase from Burkholderia cepacia by PEG/Phosphate ATPS and its biochemical characterization

This work aimed to study the partitioning of a lipase produced by Burkholderia cepacia in PEG/Phosphate aqueous two phase system (ATPS) and its characterization. Lipase was produced by B. cepacia strains in a fermenter. Enzyme partitioning occurred at pH 6.0 and 8.0, using PEG 1500 and 6000 on two tie lines. Metal ions, pH and temperature effects on enzyme activity were evaluated. Five milliliter of 7.5% olive oil emulsion with 2.5% gumarabic in 0.1M sodium phosphate buffer at pH 8.0 and 37ºC were used for the activity determinations. Results showed that crude stratum from B. cepacia was partitioned by PEG1500/phosphate ATPS at pH 6.0 or 8.0 for, which the partitioning coefficients were 108-and 209-folds. Lipase presented optimal activity conditions at 37ºC and pH 8.0; it showed pH-stability for 4 h of incubation at different pH values at 37ºC. Metal ions such as Mn2+ , Co2+, I-and Ca2+ sustained enzymatic activities; however, it was inhibited by the presence of Fe2+, Hg2+ and Al3+ . Km and Vmax values were 0.258 U/mg and 43.90 g/L, respectively. A molecular weight of 33 kDa and an isoelectric point at pH 5.0 were determined by SDS-PAGE and IFS electrophoresis, respectively.

The effect of pH on bromelain partition from Ananas comosus by PEG4000/phosphate ATPS

In this work, the thermodynamic equilibrium and applying of PEG4000/Phosphate ATPS on the purification of bromelain extracted from pineapple was studied. A rigorous study of the equilibrium curves and tie-line length from PEG4000/phosphate ATPS were done for the pH 6-11 at 25ºC. Results showed that there was augment in the PEG and salt contents with the high pH value from PEG4000/Phosphate ATPS and two-phase formation needed only increasing the PEG content. Two tie-line length at pH 11 from PEG4000/Phosphate ATPS were optimal condition for bromelain purification, one on composition of 14 percent PEG and 13 percent salt and other at 12.6 percent PEG and 12.2 percent salt, while a 25-62 folds of enzyme was found. SDS-PAGE electrophoreses had one band only, which showed that bromelain was purified. Optimum conditions of bromelain use were found at pH 7 and between 30-40ºC.

Partition of glucose oxidase from Aspergillus niger in aqueous two-phase systems based on salt and polyethylene glycol

The aim of this work was to study the isolation of glucose oxidase (GOx) from Aspergillus niger in aqueous two phase system consisting of PEG 7500 (150g l-1), potassium phosphate (175 g l-1, K2HPO4 +KH2PO4) and glucose (10 gl-1), the enzyme was partitioned in polymer phase. By sequential extraction GOx (69.2 percent) was recovered in polymer phase by 11.8 fold purification, giving a yield of 129U mg protein-¹.

Produção e extração de ácido clavulânico de Streptomyces spp. por fermentação extrativa utilizando sistemas de duas fases aquo / Production and extraction of clavulanic acid from Streptomyces spp. by extractive fermentation using aqueous two-phase system

O ácido clavulânico (AC) é um potente inibidor de β-lactamases utilizado na área médica. Métodos alternativos, econômicos e simples para sua purificação são de grande interesse. Este trabalho objetivou produzir e extrair AC de Streptomyces spp. por fermentação extrativa utilizando sistema de duas fases aquosas (SDFA) - polietileno glicol (PEG)/sais fosfato. Foi selecionado o melhor produtor de AC entre sete linhagens de Streptomyces spp. Avaliou-se a influência de cinco fatores no cultivo do melhor produtor em frascos agitados (pH, temperatura, velocidade de agitação, concentrações das fontes de nitrogênio e de carbono), utilizando planejamento experimental estatístico. Definidas as melhores condições de cultivo, foram estudadas a produção e a extração do AC em fermentação extrativa utilizando SDFA em frascos agitados e em sistema descontínuo utilizando biorreator. Em biorreator também foram realizados o estudo termodinâmico do processo de fermentação nas condições ótimas obtidas nas etapas anteriores e a determinação do coeficiente volumétrico de transferência de massa (kLa), comparando os sistemas de fermentação no meio de cultivo simples (SF) e fermentação extrativa utilizando sistema SDFA PEG/sais fosfato (SFE) sem e com crescimento microbiano. A linhagem de Streptomyces selecionada como a melhor produtora de AC foi a DAUFPE 3060, a qual apresentou a maior produção desse inibidor, 494 mg/L em 48h, em frascos agitados nas condições: pH 6,0, 32ºC, 150 rpm, 5 g/L de glicerol e 20 g/L de farinha de soja. Após a etapa de otimização realizada para o estudo da temperatura e da concentração de farinha de soja, variáveis mais significativas no estudo de seleção, a temperatura e a concentração de farinha de soja ótimas, foram 32ºC e 40 g/L, respectivamente, com produção de 629 mg/L de AC em 48h. O estudo termodinâmico confirmou que a temperatura de 32ºC é a máxima de produção do AC; após esse valor, inicia-se, gradualmente, a degradação do AC...

Clavulanic acid (CA) is a potent inhibitor of β-lactamases used in the medical field. Alternative methods, economic and simple purification are of great interest. This PhD project aims to produce and extract clavulanic acid of Streptomyces spp. By extractive fermentation using aqueous two-phase system (ATPS) - Polyethylene glycol (PEG)/phosphate salts. The best producer of clavulanic acid among seven strains of Streptomyces spp was selected. The influence of five factors in the cultivation of the best producer in flasks (pH, temperature, agitation velocity, concentrations of nitrogen and carbon sources) using statistical experimental design was evaluated. Defined the best cultivation conditions, the production and extraction of clavulanic acid by extractive fermentation using ATPS in flasks and in a batch system using a bioreactor was analyzed. In batch system using a bioreactor were also carried out the thermodynamic study of the fermentation process in optimum conditions determined in previous steps and also determined the volumetric mass transfer coefficient (kLa) comparing the fermentation systems in simple culture medium (SF) and in a extractive fermentation using aqueous two-phase system (ATPS) PEG/phosphate salts (SEF) medium with and without microbial growth. A strain of Streptomyces spp. selected as the best producer of AC was DAUFPE 3060, which showed the highest production of this inhibitor, 494 mg/L at 48h, in flasks under the conditions of pH 6.0, 32 ºC, 150 rpm, 5 g/L of glycerol and 20 g/L of soybean flour. After the optimization step, the most significant variables in the study selection, temperature and concentration of soybean flour, were studied. The optimal values were 32 ºC and 40 g/L of temperature and soybean flour concentration, respectively, with production of 629 mg/L of CA after 48h of cultivation. The thermodynamic study confirmed that 32 ºC is the maximum temperature production of CA, after this value, starts…

Aqueous Polymer Two-phase Partition for The Proteomic Analysis of Plasma Membrane From Rat Dorsal Root Ganglion Neurons / 生物化学与生物物理进展

Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. To comprehensively identify proteins of plasma membrane (PM) from small amount of dorsal root ganglion (DRG) neurons, a proteomics strategy that utilizes aqueous polymer two-phase partition in combination with differential velocity centrifugation was adopted to enrich the PM, followed by SDS-PAGE, CapLC-MS/MS and bioinformatics analysis. Western blot analysis showed that the concentration of PM in purified plasma membrane(PPM) was 2.3 times higher than that in crude plasma membrane(CPM), 15 times higher than that in whole tissue lysate (WTL). By searching against the rat IPI protein sequence database, a total of 729 non-redundant proteins were identified from the PM preparation, of which 547 had a gene ontology (GO) annotation indicating a cellular component, and 159 (21.8%) were unambiguously identified as PM proteins. A data set of plasma membrane proteins of DRG as well as a tool to study PM proteins were provided in a small amounts of sample.

Partitioning optimization of proteins from Zea mays malt in ATPS PEG 6000/CaCl2

This work aimed to establish the relationship between the compositions and pH of ATPS PEG 6000/CaCl2 and the proteins partition from maize malt and also to simplify the process optimization in ATPS for a statistical model, established by response surface methodology (RSM). Results showed that these were no influence of pH on the phase diagrams and on the composition of tie line length of PEG 6000/CaCl2 ATPS. SRM analyses showed that elevated pH and larger tie line length were the best conditions for recovering of maize malt proteins. The maximum partition coefficient by PEG 6000/CaCl2 ATPS was about 4.2 and was achieved in ATPS in a single purification step. The theoretical maximum partition coefficient was between 4.1-4.3. The process was very suitable for continuous aqueous two-phase purification due to the stability of proteins (e.g. and -amylases) and could increase their content into middle.

Este trabalho objetivou encontrar uma relação entre a composição e o pH do sistema bifásico aquoso (SBA) PEG 6000/CaCl2 e a partição de proteínas do malte de milho, e assim simplificando a otimização do processo por um modelo estatístico, estabelecido por metodologia de superfície de resposta (RSM). Os resultados mostraram que não houve influência do pH sobre os diagramas de fases e sobre a composição das linhas de amarração do SBA PEG/CaCl2. As analises RSM mostraram que em pH elevado e nas maiores linha de amarração encontra-se a melhor condição para a recuperação das proteínas do malte de milho. O coeficiente de partição máximo foi cerca de 4,2 para uma única etapa de purificação no SBA 6000/CaCl2. O coeficiente de partição máximo encontrado teoricamente esteve entre 4,1-4,3. O processo é adequado para a purificação contínua via sistemas bifásicos aquosos, já que as proteínas do malte (ex: e -amilases) são estáveis e podendo elevar sua concentração no meio.

Study on Optimization of Extraction Technology of Flavonoids from Glechoma longituba by the Integration of Aqueous Two-Phase Partitioning with Ultrasonic Extraction / 中国药房

OBJECTIVE:To optimize extraction technology of flavonoids from Glechoma longituba by aqueous two-phase partitioning with ultrasonic extraction.METHODS:The extraction technology of flavonoids from G.longituba were optimized by orthogonal experiment with ultrasonic extraction time,the volume fraction of propanol,the ratio of solid to solution,amount of(NH4)2SO4 as factors and with extraction rate of flavonoids from G.longituba as index.RESULTS:The optimal conditions were as follows:extracting for 30 min with ultrasonic wave,the volume fraction of propanol 50%,the ratio of solid to solution 1:30,and the amount of(NH4)2SO4 30%.CONCLUSION:Established extraction technology which is characterized with simple operation and mild conditions shows great advantage.

Phase Transfer Bioconversion of Penicillin G into 6-APA by Immobilized Penicillin Acylase in Recycling Aqueous Two-phase Systems With Light-pH Sensitive Copolymers / 中国生物工程杂志

Immobilized penicillin acylase was used for bioconversion of penicillin G into 6-APA in aqueous two-phase systems consisted of a light-sensitive polymer PNBC and a pH-sensitive polymer PADB.Partition coefficients of 6-APA was found to be:about 5.78,in the presence of 1% NaCl.Enzyme kinetic showed that reaction reached equilibrium at 7h or so.The 6-APA mole yields were 85.3%(pH 7.8,and 20 ℃) and this value was about 20%higher than control in reaction of single aqueous phase buffer.Partition coefficient of penicillin G(Na) washardly changeable,while partition coefficient of product,6-APA and phenylacetate acid was significantly changeable.Reason is due to Donnan effect of phase systems andhydrophobicity of products.The change of partition coefficients of products also affects bioconversion yield of products.In the aqueous two-phase systems,substrate,penicillin G,products 6-APA and phenylacetate acid are biased in top phase,while immobilized penicillin acylase is completely partitioned in bottom.Substrate,penicillin G enters into bottom phase,and it is catalyzed into 6-APA and phenylacetate acid,then the products enter into top phase.Finally,inhibition of substrate and products is removed to result in improvement of products yield.Moreover,immobilized enzymehashigher efficiency than immobilized cells and occupy smaller volume.Comparing with free enzyme,immobilized enzymehashigher stability,longer use life,completely partitioned in bottom phase and recycle.Bioconversion in two-phase systems using immobilized penicillin acylase showed outstanding advantage.The light-sensitive copolymer forming aqueous two-phase systems could be recovered by laser radiation at 488 nm or filtrated 450 nm light,while pH-sensitive polymer PADB could be recovered by isoelectric point(pH 4.1).The recovery of the two copolymers was 95%~99%.

The Application of Countercurrent Chromatography with Aqueous Two Phase System in the Separation of Proteins / 中国生物工程杂志

Aqueous two phase system(ATPS) provides a gentle, non-denaturing separation environment for proteins, enzymes. While high-speed countercurrent chromatography (HSCCC) is a liquid-liquid partition chromatography which uses centrifugal force to hold the stationary phase and facilities the mobile phase partitioning through the stationary phase, it can produce high separation efficiency with large sample loading capacity. However, the ordinary HSCCC apparatus (Type J ) fails to retention a satisfactory stationary phase of ATPS because of its high viscosity and low interfacial tension. Nevertheless, the later designed cross-axis planetary centrifuge system(X-CPC) can produce a greater lateral force field and enhances significantly the retention of the ATPS stationary phase. A review of the application of these CCC techniques with ATPS in the separation of proteins was given. Meanwhile, new techniques such as pH-peak focusing CCC and dye-ligand affinity CCC and some new CCC column design for improvement of separation efficiency and retention of ATPS stationary phase are introduced.

The separation of baicalin by aqueous two-phase extraction system / 中成药

AIM:To explore the extraction mechanism of the separation-purification of baicalin in aqueous two-phase systems. METHODS:By experiment,nonionic surfactant PEG-K_2PHO_4-H_2O two aqueous phase system was selected to phase-forming conditions,and the effect of temperature,pH,salt composition on partition of baicalin PEG/salt system. RESULTS:The extraction rate of baicalin in aqueous two-phase system was up to 98.6%. CONCLUSION:The two aqueous phase system for baicalin extraction is easy to operate has good reproductivity,and can be applied in mass production.