Application of Deep Eutectic Solvents in Extraction and Preparation of Active Ingredient of Scutellaria Baicalensis Georgi
Supervisor:zhang li wei
Deep eutectic solvents(DESs)are homogeneous stable systems formed by the interaction of hydrogen bond acceptors and hydrogen bond donors at molar ratio.DESs have many advantages,e.g.biodegradability,low toxicity,easy preparation,high solubilization strength and other novel properties.Because of these great properties,DESs have been extensively reported to extract and separate active ingredients from plants.The ultrahigh pressure extraction(UPE)is a novel and environmental extraction technology in recent years.It uses the effects of high pressure on solvent and cell structure to extract the active ingredients,which is operated under room temperature to ensure that the active ingredients will not be lost.It has the advantages of short extraction time,high extraction yield,no pollution and less impurities.Based on the development prospect of UPE and the advantages of DESs,baicalin and baicalein,as the main active components of Scutellaria baicalensis Georgi,were selected as the experimental object in this paper.The extraction effect of baicalin with DESs solvent system assisted by ultra-high pressure and microwave extraction was studied;A simple and rapid method for preparation of veterinary compound Shuang huang lian oral liquid was developed by using DESs coupled with UPE,and its toxicity was also preliminarily studied.In addition,Preparation of baicalein by enzymatic hydrolysis from DESs system was studied.The main work of this paper is as follows:(1)Nine kinds of hydrophobic DESs consisting of decanoic acid and different quaternary ammonium salts were synthesized,the effect of microwave-assisted DESs on the extraction yield of baicalin and the extraction mechanism were discussed.First,Dec A:N4444-Cl(1:2)was selected as the most appropriate DESs by comparing the extraction yield of different DESs.Based on the extraction yield of baicalin,single factor and response surface methodology(RSM)were employed to model andoptimize the parameters(extraction temperature,liquid-solid ratio,and extraction time).Furthermore,the maximum yield was achieved under optimum conditions in DES-containing aqueous solutions(33% water content),extraction temperature of 85℃,extraction time of 10 min and a liquid-solid ratio of 22.74 m L/g,which was even better than using the pharmacopoeia procedure.(2)Five kinds of choline chloride-based DESs with UPE were applied to quickly extract baicalin from Scutellaria Baicalensis Georgi.With the extraction yield of baicalin as the evaluation index,the optimal solvent was selected first,and then the extraction protocol was optimized by with the response surface methodology(RSM)by considering the impacts of DESs concentration,extraction pressure,extraction time and solid/liquid ratio on the extraction yield.Finally the optimum extraction conditions were determined.Under the optimal condition(40% water content,extraction pressure of 400 MPa,extraction time of 4 min and a liquid-solid ratio of 110 m L/g,the maximum yield of 116.8 mg/g was achieved,higher than that by the traditional extraction method.(3)A highly efficient extraction method for Preparation of veterinary Shuang huang lian oral liquid was developed by using Ch Cl-LA(1:1)coupled with UPE.Based on the best extraction yield of baicalin by Ch Cl-LA,the extraction yield of baicalin,forsythiaside and chlorogenic acid as an evaluation index,orthogonal test was applied to optimizing the the preparation conditions of Shuang huang lian preparations(prescription composition: Forsythia suspensa,Scutellaria baicalensis and Honeysuckle).The optimal conditions were determined to be 1:8 for solid-liquid ratio,400 MPa for extraction pressure,4min for extraction time,and 30% for water content,the extraction yield of chlorogenic acid,baicalin,forsythiaside reached 29.77mg/g、112.66mg/g、5.99mg/g respectively.(4)The acute toxicity of Ch Cl-LA(1:1)and the veterinary compound Shuang huang lian was preliminarily studied.The toxicity of DES wasanalyzed by acute toxicity test using Ch Cl-LA(1:1)as the test solution,and Ch Cl-LA(1:1)was administered orally using gavage to mice.The median lethal dose(LD50)was estimated though the Karber method.As a result,the estimated LD50 was 3538 mg/kg with a 95% confidence interval of3254-3855 mg/kg for oral administration.It indicated that Ch Cl-LA(1:1)is low-toxic.After the short-term administration of the Shuang huang lian oral preparation,there was no significant effect on body weight of mice,organ coefficient of mice and bio-chemical indexes of blood.(5)The optimial technology for autogenesis—enzyme hydrolysis of baicalin in choline chloride-based DESs was studied.With the hydrolysis rate of baicalin as an index,the optimum DES for baicalin hydrolysis was selected.The effects of temperature,substrate concentration and enzyme dosage on the hydrolysis rate of baicalin were investigated by single factor test and orthogonal test,and the optimum enzymatic hydrolysis conditions were optimized.As a result,the water content of 80% in Ch Cl-U(1:1)was chosen as the best solvent,the better optimization of technology were temperature 50℃,160 μL enzymes were added to each milliliter reaction system,concentration of substrate 0.2 mg/m L.The hydrolysis rate of baicalin was 92.3%,and the purity of baicalein was 82.8%.