Extraction,Purification And Primary Structure Of Polysaccharides From Trifolium Repens L.

White clover(Trifolium repens L.)is a perennial herb of Leguminosae,which is a kind of nutrient-rich pasture with high yield and wide distribution in the world.It is often used to feed livestock and build lawns.Its whole plant can also be used as medicine and has the effect of clearing heat,calming the heart and cooling the blood.Polysaccharides,a kind of natural macromolecules,are widely present in organisms and have many biological activities,such as anti-tumor,anti-oxidation,antihyperglycemic and antilipemic.In this experiment,in order to determine the most suitable extraction conditions of polysaccharides from Trifolium repens L.(TRPs),white clover was used as the raw material to extract TRPs.In addition,the crude polysaccharides were separated and purified.The primary structure of purified polysaccharides was analyzed.The experimental results are as follows:(1)TRPs were extracted by hot water extraction(HWE),ultrasonic-assisted extraction(UAE),enzyme-assisted extraction(EAE)and enzyme-ultrasonic-assisted extraction(UEAE),and four polysaccharides,namely HWE-TRPs,UAE-TRPs,EAE-TRPs and UEAE-TRPs were obtained respectively.Effects of the four extraction methods on the yield,physicochemical properties,antioxidant activity and hypoglycemic activity of polysaccharides were determined.The results showed that the yields of EAE-TRPs and UEAE-TRPs were significantly higher than that of UAE-TRPs and HWE-TRPs(P<0.05).EAE-TRPs had higher uronic acid content,smaller molecular weight,stronger antioxidant activity and significant inhibitory activity of ?-glucosidase.(2)EAE was selected to extract TRPs based on the above results and the extraction conditions were optimized.The single factor experiment was carried out first,and the extraction conditions were optimized by the response surface.The optimal extraction process was determined as follows: the liquid–solid ratio was 30 mL/g.The enzymolysis time was 87 min.The enzyme-complex dosage was 1.6%,and the pH was 6.The yield of TRPs under the conditions was 13.15±0.12%.(3)The polysaccharides were dried by three methods: hot air drying(HD),freeze drying(FD)and vacuum drying(VD).Three polysaccharides of HD-TRPs,FD-TRPs and VD-TRPs were obtained.The effects of different drying methods on the yield,physicochemical properties and activities of polysaccharides were studied.The results showed that the drying method had a significant effect on the yield of polysaccharides(P<0.05),and the yield of FD-TRPs was the highest.The total sugar content and uronic acid content of FD-TRPs were significantly higher than those of VD-TRPs and HD-TRPs(P<0.05).FD-TRPs had lower molecular weight,higher antioxidant activity and stronger inhibitory activity against ?-glucosidase.(4)The purified polysaccharide TRPs-1.2 was obtained by DEAE-52 cellulose ion exchange column chromatography and Sepharose CL-6B gel column chromatography.The primary structure of TRPs-1.2 was studied.According to the results of molecular weight distribution,TRPs-1.2 was a homogeneous fraction with a molecular weight of 4387.58 Da.The composition of TRPs-1.2 monosaccharide was determined by HPLC.It was found that TRPs-1.2 was composed of galacturonic acid,glucose,galactose and arabinose,and the molar ratio was 1.00:10.77:4.41:4.09.UV spectra revealed that TRPs-1.2 contained no nucleic acids and proteins.The results of FT-IR spectroscopy showed that TRPs-1.2 had a typical polysaccharides absorption peak.This study has developed a new utilization field of white clover.In the future,we can conduct more in-depth research on this basis,and apply white clover polysaccharides to food development,feed addition and other fields.