| Aqueous two-phase extraction system is a new and promising liquid-liquid extraction technology for extraction and purification of biomolecule.Due to the mild operation condition,the simple equipment,the low cost and the easy standard operation,the technology has been widely used in many fields,such as food,pharmaceutical and biotechnology industry.Anthraquinones are the main effective components in rhubarb,the Chinese medicine.The clinical study confirmed that the anthraquinones of rhubarb have a variety of biological activities,e.g.anti-tumor,anti-inflammatory,anti-virus,antibacterial and diarrhea.The demand for the safe active ingredient plant is increasing year by year.So how to extract anthraquinones effectively from rhubarb has an important effect on the application which is in the term of new drugs’ development,health care drug,food and cosmetics industry.This topic constructs the small molecule alcohol/salt aqueous two-phase system(ATPS)and researches into the phase diagram of ATPS and discusses the factors that influence the separation of phase,which provide a scientific basis for the further application of ATPS.Propanol/sodium sulfate aqueous two-phase system is used to extract the free anthraquinones from Rheum palmatum L..Furthermore,hydrolyzing the bond anthraquinones by adding acid improve the extraction yield of total anthraquinones greatly and separating water-soluble impurities efficaciously is realized simultaneously.At last,the antimicrobial activity of substances extracted by ATPS was analyzed in the end.The main research results were as follows:1.At first,the small molecule alcohols and inorganic salts were screened out to form ATPS.Then the nature of their phase diagram was studied,including binodal curves and tie-lines.The binodal data were determined by the turbidity titration method and the optimal equation for fitting the binodal data of small molecule alcohol/salt ATPS was selected.According to the leverage and fitting parameters,MATLAB can calculate the tie-line data of ATPS.The results show that methanol cannot form ATPS with these eight salts and ethanol and isopropanol can form ATPS with some inorganic salts,while 1-propanol can form ATPS with these eight inorganic salts.The empirical equation suitable for binodal data fitting of the small molecule alcohol/salt aqueous two-phase system was w1=exp(a+bw20.5+cw2+dw22).2.The effects of alcohol,salt and acid on small molecule alcohol/salt aqueous two-phase system were investigated.The results suggested that the order of the phase capacity is 1-propanol>2-propanol>ethanol,Na2SO4>(NH4)2SO4>NaCl.The increase in the amount of alcohol was beneficial to the phase-separation.When the concentration of salt and total volume were invariable,the volume of the top phase versus the volume of alcohols added was found to be linear.When the concentration of salt was increased,the volume of top phase was decreased.When the volume of alcohol and system were invariable,the volume of the top phase versus the concentration of salt was found to be linear.On the strong acid condition,the volume top phase decreased and then increased and finally changed into a single phase.3.Using the small molecular alcohol/salt aqueous two-phase,free anthraquinones can be extract from Rheum palmatum L..The effects of different parameters such as alcohol concentration,the kind of salt,salt concentration,temperature,time,power of supersonic and liquid/solid ratio on the yield of free anthraquinones have been discussed.The results confirm that the 1-propanol/Na2SO4 systems is better than others on the extraction yield.In the investigating range,the maximum yield of free anthraquinones(11.98 mg/g)was obtained at 50%concentration of alcohol,12%concentration of sodium sulfate,50℃,560 W,50 min and 80 mL/g.4.This thesis build a one-step method of performing extraction and hydrolysis at the same time by adding strong acid in the 1-propanol/Na2SO4 aqueous two-phase systems.The effects of different parameters such as the kind of acid,the volume of acid,temperature,time,power of supersonic and liquid/solid ratio on the yield of total anthraquinones have been researched.Furthermore,an experimental design of a three level three factors box-behnken design response surface methodology(BBD)was used to evaluate the influence of the extraction conditions on the yield of total anthraquinones and the optimum extraction conditions were optimized.The results indicate that the hydrolytic effect of H2SO4 is better than HCl and the yield of total anthraquinones was raised by 1.5 times.The order of influence on yield is temperature>time>liquid/solid ratio.The optimal experimental parameters were 75℃,30.00 min,20.00 mL/g.The predicted yield of total anthraquinones was 20.03 mg/g.The practical yield(19.59 mg/g)was similar to predicted value and it confirmed the model that was stable and reliable.The model is able to predict experimental results of extraction yield of total anthraquinones.5.One-step method was compared with usual method which extracted anthraquinones highly,usual solvents(methanol/ethanol)and two-step method of extraction and hydrolysis.The results indicated that the one-step method had higher yield and shorter time.The 1-propanol/Na2SO4 ATPS also could remove some sugars to separate roughly.The product could not need re-extraction and the solvents of ATPS could be reused.So procedure of one-step method is briefer,lower cost,and greener.6.Filter paper disc method was used to determine the antibacterial activity of extraction by one-step method for Staphyloccocus aureus,Pseudomonas aeruginosa,Bacillus subtilis and Escherichia coli.Double dilution method was used to obtain the minimum inhibitory concentrations(MIC)and minimal bactericidal concentrations(MBC).The results showed that the extraction had inhibitory effect on the four kinds of bacteria,and the order is Staphyloccocus aureus>Pseudomonas aeruginosa>Bacillus subtilis>Escherichia coli.The extraction had the strongest inhibitory effect on Staphyloccocus aureus and the MIC was 4.688 mg/mL and the MBC was 9.375 mg/mL. |
PDF Full Text Request