Extraction And Purification,Structure Identification And Biological Activities Of Polysaccharide From Pumpkin Seeds

Pumpkin seeds are ripe seed of pumpkin.Pumpkin seeds were rich in proteins,essential fatty acids,sterols,arginine,vitamins and trace elements.Pumpkin seeds have been implicated in providing many health benefits such as lungs,phlegm,eliminating pain and diuretic effects.So far,there are few reports on the research of pumpkin seeds polysaccharides.In order to further improve the utilization of pumpkin seeds and play its potential economic value,a series of studies of pumpkin seeds polysaccharides were carried out in this paper.The present study reported extraction,purification functional properties,structural identification and biological activity of pumpkin seeds polysaccharides.An enzyme-assisted procedure for the extraction of the water-soluble polysaccharides from pumpkin seeds was investigated using response surface methodology.Under the optimal conditions,the PSP yield was 3.22±0.04%.Response surface methodology and the Box-Behnken design based on single-factor experiments were applied to optimize the deproteinization conditions.Under the optimal deproteinization conditions,the rate of deproteinization was 32.77%±1.23%.Functional properties showed that water holding capacity and oil holding capacity increased with temperature.The solubility was determined at different temperatures?25,45 and 65°C?,increasing when these variables were increased.Emulsifying ability increased and emulsifying stability decreased when the weight of powder of pumpkin seeds polysaccharides to oil volume ratio was increased.In addition,the pumpkin seeds polysaccharides showed good cation exchange capacity.With successive separation by DEAE cellulose DE-23 ion exchange column chromatography and Sephadex G-25 gel filtration chromatography,a water-soluble polysaccharide,PSP-1,was obtained.The determination of physicochemical properties showed that PSP-1 was insoluble in organic solvents such as ethanol,ether and acetone,the contents of sugar and protein were 86.43%and 7.85%respectively,excluding uronic acid,polyphenols and starch.Thermal analysis showed that PSP-1 has better thermal stability when the temperature is lower than 150°C.PSP-1 showed only one single and symmetrical peak on HPGPC,indicating that PSP-1 is a homogeneous polysaccharide.The average molecular weight of PSP-1 was estimated to be about 3728 g/mol.The monosaccharide composition analysis showed that PSP-1 consists of mannose,glucose and galactose in a molar ratio of 1.00:4.26:5.78.The results of automatic amino acid analyzer showed that the PSP-1 contained 16 general amino acids and was rich in glutamic acid?3.14?g/mg?,leucine?2.79?g/mg?and proline?2.54?g/mg?.The alkali sensitive O-glycoprotein was identified by?-elimination reaction.IR spectrum indicated the PSP-1 had absorptions peaks of pyranose,?-type and?-type glycosidic without uronic acid.Periodic acid oxidation,1D NMR and2D NMR analysis showed that the backbone of PSP-1 consisted of?1?6?-linked-?-D-galactopyranose,?1?6?-linked-?-D-glucopyranosyl and?1?3,6?-?-D-mannopyranosyl.Branch linkages were composed of?-D-glucopyranosy and?1?4?-linked-?-D-galactopyranose.The results of Congo red test showed that the PSP-1 had no three helical structure.PSP-1 has a certain scavenging ability for DPPH,·OH and O₂^-·radicals and nitrite ions,and has good anti-lipid peroxidation ability.PSP-1 has a certain inhibitory effect on?-glucosidase and?-amylase,and?-amylase inhibition mechanism is achieved through reversible competitive inhibition.In addition,there was no significant change for molecular weight of PSP-1 during the digestion of simulated gastric fluid and intestinal fluid,indicating that PSP-1 may not be digested and decomposed by the simulated stomach and small intestine.