Extraction And Bioactivity Of Polysaccharides From Dendrobium Officinale By Deep Eutectic Solvents

Dendrobium officinale is a rare Chinese traditional medicine,and it is extremely strict with growth conditions.Its natural resources are increasingly scarce cuased by the low natural reproductive ability and the increasing market demand.The main active ingredient of D.officinale is polysaccharides.At present,the main extraction method for extracting D.officinale polysaccharides is water extraction and precipitation by alcohol,but it suffers from shortcomings such as high temperature and low efficiency.This paper aimed to use new solvent-deep eutectic solvents?DESs?extract D.officinale polysaccharides and optimize the condictions of purifying polysaccharides.The structure and antioxidant activity of the purified polysaccharides are studied.The main contents are as follows:?1?19 kinds of DESs including alcohols,amides,organic acids,and halogenated metal salts as hydrogen bond donors or acceptors were successfully synthesized.The water solvent was used as a control group,and the solvent direct extraction method was compared with the compound enzymes?cellulose:pectinase=1:1?assisted extraction of D.officinale polysaccharides,DESs were superior to water extraction,and complex enzyme-assisted extraction was superior to solvent direct extraction.The yield of polysaccharide extracted by ChCl/Lev and complex enzymes-assisted ChCl/Gly is about 40%,44%,respectively.And the water ectraction is about 37%.Finally,response surface optimization was performed using ChCl/Lev solvent and compound enzyme-assisted ChCl/Gly extraction method.?2?Three types of macroporous resins S-8,AB-8 and H1020 were compared with activate carbon,the decolorization rate,deproteination rate,and polysaccharides recovery rate of DES extracts purified with macroporous resin are increased to 30%,60%and 10%,respectively.Two fractions of polysaccharides?DOP1-DES,DOP2-DES?are obtained after fractionated using Sephadex G75 and Sephadex G200,and the purity and molecular weight?Mw?of DOP1-DES and DOP2-DES were 96.78%,97.37%and 297911 Da,30400 Da,respectively.The purity of the crude polysaccharides CDOP-DES extracted by DES solvent was 92.25%,and the purity of water extracting polysaccharides CDOP was 87.69%.?3?The monosaccharide composition of DOP1-DES and DOP2-DES polysaccharides were glucose and mannose with the ratios of 2.2:1 and 3.7:1 respectively.The results of FTIR showe that DOP1-DES and DOP2-DES have peaks of vibration with specific vibrational absorption peaks belonged to glucose and mannose.NMR and methylation analysis show the linking pattern of DOP1-DES are?1,4?-linked?-D-Glcp,?1,6?-Linked?-D-Glcp and?1,3,6?-linked?-D-Manp.The linking of DOP2-DES are?1,4?-linked?-D-Glcp and?1,3?-linked?-D-Manp,?1,6?-linked?-D-Glcp and?1,3,6?-linked?-D-Manp.The sulfuric acid content of DOP1-DES and DOP2-DES is determined to be 8.22±0.27%and 7.47±0.32%,respectively.After I₂-KI reaction prove that DOP1-DES and DOP2-DES do not contain starch and may contain more short-chain branches.After Congo red test,it is proved that DOP1-DES does not have obvious triple helical conformation,and DOP2-DES has a certain helical conformation.?4?The bioactivities were investigated.The effect of different extraction methods,extraction solvents,and different molecular weights are investigated on antioxidant activeties.The results show that polysaccharide extracted by enzyme-assisted DES method was superior to water extraction,The CDOP-DES,DOP1-DES and DOP2-DES were showed different ancioxidant activities.And the polysaccharides obtained by different extraction methods have different antioxidant activities.In addition,the effect of enhancing immunity of polysaccharides was determined by in vitro experiments.A cell model was constructed by RAW264.7 to measured that the secretion of NO,TNF-?and IL-6.The results indicating that polysaccharide has the effect of promoting immunity.Enzyme-assisted DES extraction can obtain higher yield of polysaccharides,the extraction conditions are mild.Therefore,it is easy to realize industrial production.Structure identification of purified polysaccharides after purified by macroporous resin and glucan gel provides a reliable structure for further study on the structure-activity relationship of D.officinale polysaccharides,and used to guide the chemical modification of the polysaccharide structure such as thioation,phosphorylation in order to enhance the biological activity,and as a reliable crude drug quality identification indicators.