Study On Field-intensified-assisted Extraction Coulped With Multi-phase Aqueous Extraction For Separation Of The Effective Constituents From Euonymus Alatus

Euonymus alatus is the dried winged branch or its attachments of Euonymus alatus?Thunb.?Sieb.belonging to Celatraeeae family.So far Euonymus alatus has been used as C hinese folk remedies for over 2000 years.Recent researches showed that its extracts have antihypoglycemic,antihypertensive and antitumor activities etc.The main constitutions of Euonymus alatus are flavonoids and phenolic acids,which were often extracted by the conventional methods of heat-reflux and cold soaking extraction.However,these methods are not only reagent-consuming or inefficient,but also harmful to the environment.In this paper,the effects of different molecular weight of polyethylene glycol?PEG?,the composition of phase components and temperature on phase equilibria and phase transition law were systematically investigated according to physico-chemical properties.In terms of the chemical properties of the effective constituents in Euonymus alatus,they were extracted by a homogeneous and/or biphasic system of the PEG aqueous solution based on homogeneity and demixing effect under ultrasound and microwave fields.For the purpose of quantitative analysis,the objective of our study is to develop a simple,rapid,efficient sample pretreatment method for extraction,separation and enrichment of target compounds.The main studies and results in this paper were as follows:1.The main constituents in Euonymus alatus were qualitatively analyzed by ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry?UPLC-QTOF-MS?.Orientation,catechin,dihydromyricetin,gallic acid and ellagic acid were identified by UPLC-QTOF-MS data and UV spectrum.Those compounds fragmentation pathways were deduced in detail.Flavonoids and phenolic acids mentioned above were the main effective constituents in Euonymus alatus.2.The methods to determination of catechin and dihydromyricetin from Euonymus alatus were developed by reversed high performance liquid chromatography and its total flavonoids were measured by UV.The mobile phases were consisted of acetonitrile-water containing 0.5% phosphoric acid,the detection wavelength was 280 nm for catechin and dihydromyricetin,the retention time of the two constituents were 19.09 min and 26.64 min,respectively.Their linear ranges were from 0.8992-56.20 mg×L-1 and 1.004-50.20 mg×L-1,respectively.Recoveries were between 99.5-101.5%.The RSDs were 1.91% and 0.84%,respectively;the retention time of gallic acid and ellagic acid were 6.21 min and 32.79 min,respectively.They had a good liner relationship in the range of 0.9200-46.00 mg×L-1 and 0.8580-42.90 mg×L-1,respectively.Recoveries were between 99.0-101.2%.The RSDs were 0.79% and 0.96%,respectively;Selecting rutin as a reference substance,hydrochloric acidMg reaction assay was adopted to determined the content of total flavonoids,rutin had a good liner relationship with absorbance in the range of 0.1024-1.024 mg×mL-1.Recoveries were between 99.76-101.4%.The RSD was 0.98%.Using gallic acid as a reference substance,direct spectrophotometric measurement of the total phenolic acids at 281 nm,gallic acid had a liner relationship with absorbance in the range of 0.92-18.4 mg×mL-1.Recoveries were between 99.50-100.7%.The RSD was 0.50%.These methods had already been applied to the determination of the effective constituents from Euonymus alatus,and they turned to be reliable methods.3.Liquid-liquid equilibrium of the PEG-?NH₄?₂SO₄ aqueous two-phase systems was developed in detail.Phase diagrams were determined and validated by the cloud point method and Merchuk,respectively.The effects of molar mass of PEG and temperature on the liquid-liquid equilibrium for aqueous two phase systems were sysmatically investigated.The bimodal curves were fitted and evaluated using empirical equations.The tie lines were correlated using the Othmer-Tobisa and Bancrof equations with sasmactory correlation coefficients?R2>0.99?,demonstrating the reliability of the equation.The resulted showed that the ability of PEG for two phase formation followed the order: PEG-6000 > PEG-4000 > PEG-2000 > PEG-1000 > PEG-400;Moreover,two phase area of PEG-ammonium sulfate was expanded with increase of temperature.The objects of this work are to provide the basis for their further application.4.Based on screening PEG of different molecular weight,PEG-400 was chosen to be the extraction agent.Ultrasonic-assisted solvent-reflux extraction was adopted to extract the catechin,dihydromyricetin and total flavonoids from Euonymus alatus.A response surface methodology combined with single factor optimization experiment was applied to maximize the extraction yields.The extraction and enrichment of target compounds were further investigated by temperature-induced cloud-point extraction.The optimum extraction conditions for ultrasonic-assisted extraction were: the concentration of PEG-400 was 15%?w/w?,particle size was 80 mash,solvent-tomaterial ratio was 60:1,and extraction time was 10 min.The yield of experimental extraction for total flavonoids,catechin and dihydromyricetin under the optimum extraction conditions were 3.126,0.657 and 1.353 mg/g respectively.The cloud-point extraction of flavonoids was induced after adding 5.6 g ammonium sulfate with the temperature of 75? and the solution pH of 3.5.The enrichment effect of the upper PEG-400 phase was excellent with enrichment factor of above 5.Compared to the result of ethanol as solvent,PEG-400 had better extraction selectivity.Extraction efficiency was significaltly improved by ultrasonic-assisted field,being superior to different extraction methods.5.PEG-400-?NH₄?₂SO₄ aqueous two-phase system?ATPS?was chosen to be the extraction agent through screening PEG of different molecular weight.Microwaveassisted aqueous two-phase extraction?MAATPE?was exploited to extract the phenolic acids constituents from Euonymus alatus.A response surface methodology combined with single factor experiment was applied to optimize the extraction condition.The optimum extraction conditions for MAATPE were validated according to the following: the ATPS composition of 18%?w/w??N H₄?₂SO₄ and 26%?w/w?PEG-400,extraction temperature of 95?,and solvent-to-material ratio of 60:1.The results showed that extraction yields of gallic acid and ellagic acid under the optimum extraction conditions were 0.425 and 0.1517 mg/g,respectively.Microwave-assisted field can intensify the extraction processs,improving extraction efficiency compared to other extraction methods.