Optimization Of Extraction Process Of Polygonatum Odoratum Polysaccharide By Deep Eutectic Solvents And Study On Its Activity And Structure

Polygonatum odoratum is the rhizome of Polygonatum of Liliaceae,It has the effects of Nourishing Yin,moistening dryness,generating saliva and relieving cough.Modern pharmacological studies have found that Polygonatum odoratum has the functions of antioxidation,regulating immunity,lowering blood sugar and blood pressure.Polygonatum odoratum polysaccharide is the main active component in Polygonatum odoratum and an important factor affecting the biological activity of Polygonatum odoratum.However,due to the large molecular weight and complex structure of polysaccharides compared with other active substances,the research progress of polysaccharides is slow.At present,the research on Polygonatum odoratum polysaccharide is limited to the optimization of traditional extraction methods,but there is little research on the structure and structure-activity relationship of Polygonatum odoratum polysaccharide.Moreover,the traditional extraction methods,such as hot water extraction,limit the research and development of Polygonatum odoratum polysaccharide due to the disadvantages of long time-consuming and low extraction rate.Extracting bioactive substances with deep eutectic solvents is a hot research direction in recent years.At present,bioactive substances such as polyphenols,flavonoids,alkaloids and polysaccharides have been successfully extracted by DESs.There are few reports on Extracting Polysaccharides by DESs.In this paper,DESs and hot water extraction were used to extract polysaccharides from Polygonatum odoratum,and the extraction process of DESs was optimized.The effects of extracting polygonatum odoratum polysaccharide from DESs on its physical properties and biological activities were analyzed,and Polygonatum odoratum polysaccharide was isolated and purified.The monosaccharide composition,molecular weight and structural information of the purified polysaccharide were determined.The effects of DESs extraction on the properties and structure of Polygonatum odoratum polysaccharide were analyzed to explore the relationship between structure and functional activity.The main results are as follows:(1)Five different combinations of DESs solutions were prepared as solvents to extract polysaccharides from Polygonatum odoratum.The combination of DESs with the highest extraction rate was selected for process optimization.The results showed that the extraction rate of Polygonatum odoratum polysaccharide by choline chloride urea combination DESs was the highest.The effects of conditions on the extraction rate were investigated by taking the molar ratio of choline chloride to urea,extraction time,extraction temperature,solvent water content and liquid-solid ratio as single factors.Fixed urea: choline chloride = 3:1,response surface optimization test was carried out with extraction rate as response value,extraction time,extraction temperature,solvent water content and liquid-solid ratio as single factors.The optimum extraction process was as follows: molar ratio 3:1,extraction temperature92 ℃,extraction time 41 min,solvent water content 19%,liquid-material ratio 29:1.Under this condition,the extraction rate of polysaccharide was 29.03±0.54%,about4.67 times that of hot water extraction.After four deproteinization operations,most of the protein in the polysaccharide can be removed and the polysaccharide retention rate is large.The recycling test of DESs determined that the recovered DESs could be used for polysaccharide extraction again and could be reused for at least 5 times.(2)The polysaccharides obtained by the two extraction methods were freeze-dried to obtain crude polysaccharide powder,which were named DPo P(DESs extraction of Polygonatum odoratum polysaccharide)and WPo P(hot water extraction of Polygonatum odoratum polysaccharide).Two kinds of Polygonatum odoratum polysaccharides were prepared into a solution with a certain mass concentration,and their antioxidant capacity,anti glycosylation capacity and α-Amylase inhibition,emulsification and rheological properties.The results showed that the antioxidant capacity of dpop in vitro was higher than that of wpop.Both polysaccharides have good ability to inhibit the formation of ages and α-Amylase inhibition.DPo P has better emulsifying activity,stability and apparent viscosity than WPo P.The infrared spectra of the two polysaccharides showed that they both contained polysaccharide absorption characteristic peaks and existed β-Pyranoside bond and α-Pyranoside bond.(3)DPo P and WPo P of Polygonatum odoratum polysaccharides were separated by DEAE Sepharose ion exchange column chromatography.Four components of the two polysaccharides(DPo P-A,DPo P-B,DPo P-C,DPo P-D and WPo P-A,WPo P-B,WPo P-C,WPo P-D)were obtained respectively.The molecular weight of Polygonatum odoratum polysaccharide was determined by high performance gel permeation chromatography(HPGPC).The components with large content and simple molecular weight distribution were selected for gel purification.The monosaccharide composition of purified components was determined by gas chromatography-mass spectrometry(GC-MS),the glycosidic linkage mode of Polygonatum odoratum polysaccharide was determined by methylation,and the functional group composition of purified components was determined by FTIR.The results showed that DPo P-A-1 was composed of glucose,mannose and fructose.The molar mass ratio was glucose: mannose: fructose = 0.368:0.133:0.499.WPo P-A-1,WPo P-A-2 are composed of galactose.The molar ratio of monosaccharides of WPo P-A-1 is galactose: glucose: mannose: fructose glucose,mannose and fructose=0.688:5.685:4.432:0.588.The molar ratio of monosaccharides of WPo P-A-2 is galactose: glucose: mannose: fructose glucose,mannose and fructose=0.475:6.034:2.562:0.479.The monosaccharides extracted from Polygonatum odoratum polysaccharides by DESs were less than those extracted by hot water extraction,but the monosaccharides were evenly distributed.The infrared spectra of DPo P-A-1 and WPo P-A-1 showed that both had polysaccharide characteristic peaks and pyranose.The methylation results showed that the extraction of Polygonatum odoratum polysaccharides by DESs had no effect on the glycosidic linkage mode.The molecular structure formulas of the two polysaccharides were deduced from the methylation and NMR spectra.The main glycosidic bonds of DPo P-A-1 were comprised of α-D-Glcp-1→2-β-D-Fruf-1→,→1)-β-D-Fru-(2→1,6)-β-D-Fru-(2→,→6)-β-D-Glcp-(1→1,6)-β-D-Fru-(2→,→4)-β-D-Manp-(1→1,6)-β-D-Fru-(2→,α-D-Glcp-(1→4)-β-D-Manp-(1→,α-D-Glcp-(1→6)-β-D-Glcp-(1→.The main glycosidic bonds of WPo P-A-1 were comprised ofα-D-Glcp-1→2-β-D-Fruf-1→,→1)-β-D-Fru-(2→1,6)-β-D-Fru-(2→,→4)-β-D-Glcp-(1→1,6)-β-D-Fru-(2→,→6)-β-D-Glcp-(1→1,6)-β-D-Fru-(2→,→4)-β-D-Manp-(1→1,6)-β-D-Fru-(2→,α-D-Glcp-(1→4)-β-D-Manp-(1→,α-D-Glcp-(1→4)-β-D-D-Glcp-(1→,α-D-Glcp-(1→6)-β-D-Glcp-(1→.