Research And Application Of Imidazolium-based Ionic Liquids For The Separation And Analysis Of Traditional Chinese Medicine And Protein

Ionic liquids (ILs) which are composed of organic cations and inorganic ororganic anions are famous as “novel solvents with potential salts”. They have manykinds of unique advantages including tasteless, no flammability, extremely low vaporpressure, remarkable dissolving and extracting capacity, high thermal and chemicalstability and designable structures, etc. Recent years, with the rise of the GreenChemistry concept, people are trying to reduce or eliminate the use of hazardoussubstances in chemical industry during the process of designing, testing, productingand post-processing. ILs have been used in many fields, such as organic synthesis,electrochemistry, separation and purification. Because of the advantage ofenvironment-friendly, ILs are recommended to act as extractive agents to substitutetraditional organic solvent, and getting more and more attention.Traditional Chinese medicine(TCM), which is a treasure of China, has severalthousand years of history. The research of TCM pharmacological properties has beensocial concerns for a long time. However the process of the quality control over TCMhas impeded its development in international marketplace. The extraction anddetection method is particularly urgent in order to establish an efficient, highly sensitive,and highly accurate bioactive ingredientAqueous two-phase system is consisted of two immiscible polymer solution orimmiscible salt solution with polymer solution. It is mixed by a certain concentrationof two aqueous solutions which are shaked and standing for a while, if concentrationsof exceed a determined value, a two-phase system will be generated. Aqueoustwo-phase extraction system can maintain the activity of the target composition and theoperation can be carried out continuously, it has been widely used in the field ofbiochemistry, cell biology and bio-chemical research, and it can be used for thebiotransformation, separation and purification of proteins nucleic acids and viruses.In this paper, imidazolium-based ILs were selected as extraction agent,microwave-assisted extraction, which is a modern sample pretreatment technique, wasselected as extraction and separation method. We have successfully separated andextracted the target bioactive components of kaempferol and quercetin from two kindsof medicinal herbs including Toona Sinensis and Rosa Chinensis respectively. Severaloperating parametersof extraction were optimized, and the optimal conditions had beendetermined at last. During this research, we have combined ILs with two aqueous phase extraction technology and successfully applied them to separate protein. Thisstudy was carried out as the following three aspects:1. Imidazolium-based ionic liquids microwave-assisted extraction of quercetinfrom Toona sinensisIonic liquids microwave-assisted extraction (ILs-MAE) has been developed toextract quercetin from Toona sinensis. The retention times of standard solution andsample extract were contrasted and liquid chromatography-mass spectrometry (LC-MS)were also employed to confirm that Toona sinensis do contain active ingredient ofquercetin. Several operating parameters of ILs-MAE, including the radiating time ofmicrowave, extracting temperature, solid–liquid ratio and the concentration of theoptimum ionic liquid, were successively optimized by single-factor experiments and L9(34) orthogonal array experiments. Finally, the optimum conditions of ILs-MAE wereset as follows: the sample was extracted by10ml,0.1mol/L [Bmim]Br aqueoussolution for8min, and the extraction was carried out under60℃. Besides, thesolid–liquid ratio was set at1:20. In order to find a better method for the quercetinextraction, four typical extractive methods, including ionic liquids-based macerationextraction (ILs-ME)， ionic liquids-based heating extraction (ILs-HE) and ionicliquids-based ultrasonic-assisted extraction (ILs-UAE), were selected to makecomparison with ILs-MAE. Results indicated that the amount of quercetin extracted byILs-MAE was higher than any other methods, and the amount of quercetin got byILs-MAE was182.8mg/g. In this study, reversed phase high performance liquidchromatography (RP-HPLC) with ultraviolet detection (UV-DAD) was employed forthe analysis, results indicated that the developed approach is linear betweenconcentrations from15.20mg/L to213.2mg/L of quercetin and the regressioncoefficient (R) was0.9992. The recoveries of quercetin extracted by these fourmethods were95.07-105.8%with RSDs lower than3%. Repeatability and intermediateprecision of the proposed method were both satisfactory. The mechanism of thismethod was discussed by means of kinetic mechanism, SEM and FT-IR to contrast thechange of the microstructure and chemical structure of Toona sinensis samples beforeand after different extraction procedures. At last, this method was employed to analyzereal samples and the results were satisfactory.2. Design and performance evaluation of imidazolium-based ionic liquidsmicrowave-assisted simultaneous extraction of kaempferol and quercetin from Chineseherbal plantsIn consideration of the high value, complicated preparation technology and the tedious post-processing of ILs, a more efficient method which can simultaneous extractmore than one kind of bioactive components from TCM was established, which canreduce the consumption of extracting agent, get more active ingredients by consuminglittle power.Midazolium-based ILs were selected as extraction agent and twenty types of ILshave been synthesized and investigated. An ILs-MAE technique has been successfullydeveloped for the simultaneous effective extraction of quercetin and kaempferol fromToona Sinensis and Rosa Chinensis respectively. Several significant operatingparameters of ILs-MAE were respectively optimized by a series of single factorexperiments and orthogonal design L9(34) experiments. The extraction efficiencies offour extractive methods, including ILs-ME, ILs-HE, ILs-UAE and ILs-MAE, werecontrasted. Results indicated that ILs-MAE was higher than any other methods.This work has investigated the influence of different extraction conditions on theextraction amount, the optimum conditions of ILs-MAE for Toona Sinensis were set asfollows: the sample was extracted by10ml,2mol/L [Bmim]Br aqueous solution for20min, and the extraction was carried out under60℃. Besides, the solid–liquid ratio wasset at1:30. As for the analysis of Rosa Chinensis, the optimum conditions were set asfollows: the sample was extracted by10ml,2.5mol/L [Bmim]Br aqueous solution for20min, and the extraction was carried out under70℃. Besides, the solid–liquid ratiowas set at1:40.Compared with ILs-ME, ILs-HE and ILs-UAE, the optimized approaches weredemonstrated as the optimal techniques with the highest extraction efficiency and theshortest extraction time.The results indicated that the extraction amounts of quercetin in Toona Sinensisextracted by [Bmim]Br and in Rosa Chinensis extracted by [Omim]Br were213.5mg/gand134.3mg/g. And the extraction amounts of kaempferol in Toona Sinensis extractedby [Bmim]Br and in Rosa Chinensis extracted by [Omim]Br were16.12mg/g and9.070mg/g. RP-HPLC with UV-DAD was employed for the simultaneous analysis ofkaempferol and quercetin. The developed approach for analyzing quercetin were linearbetween the concentrations from32.10mg/L to233.3mg/L in Toona Sinensis and25.70mg/L to160.5mg/L in Rosa Chinensis with the regression coefficients (R) of0.9998and0.9999. And for analyzing kaempferol, the developed approach were linearbetween the concentrations from0.73mg/L to23.20mg/L in Toona Sinensis and0.19mg/L to15.00mg/L in Rosa Chinensis with the regression coefficients (R) of0.9993and0.9996. The recoveries were95.75-106.3%with the RSDs of lower than3.00%. The LODs were1.87-2.26μg mL-1. This proposed method had good sensitivity, fineaccuracy and reproducibility, microstructures and chemical structures of these tworesearched samples before and after extraction were also investigated by FT-IR andSEM, and this method was applied to real samples with satisfactory results.3. Application of imidazolium-based ILs aqueous two-phase system in proteinextractionGreen solvents-Ils and aqueous two-phase extraction technology were combinedin this study, four kinds of water-soluble imidazole ionic liquids, including [Amim]Cl,[Pmim]Br,[Bmim]Br and [Bmim]OTM, which can form aqueous two-phase systemwith K2HPO4were selected as extraction agent. This study examined the extractionefficiency of different types of imidazole ionic liquids-aqueous two-phase system forBSA, and the result indicated that [Bmim]Br got the highest extraction efficiency.Imidazole ionic liquid aqueous two-phase system was a green and efficient method,which can be successfully applied for the separation and extraction for proteins.The phase diagrams of these four ILs were measured by turbidity titration at25℃,and the results indicated that the order of their ability is that [Bmim]OTM＞[Bmim]Br＞[Pmim]Br＞[Amim]Cl.The detection wavelength was set at278nm for BSA, whichwas the maximum UV absorption wavelength. The developed approach is linearbetween concentrations from0.3mg/mL to2.5mg/mL and the regression coefficient(R) was0.99995. Several operating parameters of imidazole-based ILs aqueoustwo-phase system in protein extraction were successively optimized by single-factorexperiments, and the optimum conditions were set as follows: the extraction time was15min, extraction temperature was30℃, K2HPO4was added in0.8g and ILs wasadded in0.4g. The methodological study results showed that the precision,reproducibility and stability of the proposed method were satisfactory. The mechanismof this method was discussed by ultraviolet spectrum detection, transmission electronmicroscopy and dynamic light scattering detection. The results show that during theextraction, the structure of protein has not been destroyed. As for the extraction power,we preliminary speculated that during the extraction process, ILs and BSA moleculeswere clustered to form a larger particle group which may be one of the engines ofaqueous two-phase system of protein extraction process.