Simultaneous Extraction Of Polysaccharides And Triterpenic Acids From Loquat Leaves By Two-phase Deep Eutectic Solvent And The Study Of Isolation And Purification

As the main by-product of loquat（Eriobotrya japonica（Thunb.）Lindl.）,loquat leaf,rich in functional bioactive components,is used as both medicine and food.Currently,the bioactive compounds of loquat leaf are usually extracted by single polar solvents,for example,water and volatile organic solvents,which have disadvantages such as low extraction efficiency,high pollution and high energy consumption.At the same time,the incomplete extraction of non-target compounds also causes a waste to some extent.In this thesis,a two-phase solvent system was developed with hydrophilic deep eutectic solvent（DES）choline chloride/ethylene glycol and hydrophobic deep eutectic solvent（HDES）thymol/benzyl alcohol.Then this two-phase solvent system was used for the simultaneous extraction of polysaccharide and triterpenic acid from loquat leaves.Purification processes based on macroporous resin column chromatography and ODS reverse-phase silica gel column chromatography were established for purification polysaccharide and triterpenic acid samples,respectively.High-purity polysaccharide and triterpenic acid samples were obtained.The antioxidant activities of the attained triterpenic acid samples from loquat leaves were investigated in vitro.The main results were as follows:（1）DES and HDES can form a two-phase solvent system under certain conditions.The two-phase solvent system can be adjusted by changing the water content of DES and the volume ratio between DES and HDES.The two-phase solvent system developed with DES of 50%water content and volume ratio of 1:1 between DES and HDES was suitable for the simultaneous extraction of polysaccharide and triterpenic acid from loquat leaves.The optimized extraction conditions were extraction time 40 min,liquid-solid ratio 25（m L/g）,extraction temperature 60°C and ultrasonic power 140 W.Under these conditions,the extraction yields of ursolic acid and polysaccharide were 15.79±0.18 mg/g and 48.96±0.56 mg/g,respectively.（2）S-8 macroporous resin showed high efficiency on the purification of crude loquat leaf polysaccharide.The optimized purification processes were sample loading volume 9 m L,sample concentration 1 mg/m L,sample loading flow rate 1 m L/min,eluent ethanol concentration 50%,and eluent flow rate 1 m L/min.Under these conditions,the polysaccharide retention rate,protein and pigment removal rates obtained by S-8macroporous resin were 51.02±1.43%,67.51±2.35%and 75.15±1.87%,respectively.The purity of the purified polysaccharide was 68.37%,which was 2.66 times of the crude polysaccharide.（3）Crude loquat leaf triterpenic acids and HDES could be recovered from HDES extracting solution through low-pressure ODS reverse-phase silica gel column chromatography,preliminarily.The purity of the crude triterpenic acid sample was 81.18%,reffering to the total contents of maslinic acid,corosolic acid,oleanolic acid and ursolic acid,with a mass ratio of 11:26:10:53.The crude triterpenic acid sample can be further separated through ODS reverse-phase silica gel column chromatography under high pressure to obtain corosolic acid sample（including maslinic acid）and ursolic acid sample（including oleanolic acid）.The purity of the isolated corosolic acid sample was 69.30%,and the mass ratio between maslinic acid and corosolic acid was 7:18;the purity of ursolic acid sample was93.94%,and the mass ratio between oleanolic acid and ursolic acid was 4:21.（4）All prepared triterpenic acid samples showed certain antioxidant activities.The IC₅₀values of the crude triterpenic acid sample,corosolic acid sample and ursolic acid sample on DPPH free radical scavenging were 0.070 mg/m L,0.025μg/m L and 0.277 mg/m L,respectively.And the IC₅₀ values on ABTS free radical scavenging ability were 0.251mg/m L,0.252 mg/m L and 1.581 mg/m L,respectively.To sum up,the two-phase solvent system developed with choline chloride/ethylene glycol and thymol/benzyl alcohol is suitable for the simultaneous extraction of polysaccharide and triterpenic acid from loquat leaves.Macroporous resin could effectively remove the impurities such as protein and pigment and improve the purity of loquat leaf polysaccharide.ODS reverse-phase silica gel can recover HDES efficiently and realize the separation and large-scale preparation of corosolic acid and ursolic acid from crude loquat leaf triterpenic acids.This study provides theoretical basis and technical support for the development and utilization of loquat leaf in the fields of medicine,health care products,functional food,cosmetics and so on,and has great significance for promoting the extension of the loquat industry chain and industrial efficiency.