Isolation,Purification,Structural Characterization And Application Of Perilla Polysaccharides

The Chinese herbal medicine and food materials in the drug and food homologous catalogue have the advantages of safety and nontoxicity.Perilla frutescens and Perilla frutescens seeds are among the catalogue.The natural products extracted from Perilla frutescens and Perilla frutescens seeds are very rich and proved to have important application value and market prospect.In this paper,perilla polysaccharides are extracted from perilla leaves(PL)and perilla seed meal(PSM).On the one hand,it can enrich the basic research and development of natural active ingredients of perilla,on the other hand,it can improve the comprehensive utilization of Perilla resources.The polysaccharides in different varieties and periods of PL and different varieties of PSM were compared.The polysaccharides in PL and PSM of different varieties and periods were compared to screen perilla varieties with high and stable polysaccharide content.Response surface methodology(RSM)was used to optimize the extraction conditions.The extraction method which is energy-saving,efficient and easy to realize industrialization was selected.The purified components of Perilla polysaccharides prepared under optimized process conditions were obtained after step-by-step purification.The components,physical and chemical properties,monosaccharide composition and ratio,thermal stability and antioxidant biological activities of Perilla polysaccharide were compared,and the Perilla polysaccharides with good antioxidant effect were selected for product development and functional verification of Perilla polysaccharides effervescent tablets.The main research contents and results of are as follows:(1)Hot water extraction(HWE)and ultrasonic assisted extraction(UAE)were used to extract perilla leaves polysaccharides(PLP)and perilla seed meal polysaccharides(PSMP).The results showed that the highest average PLP yield of 24 varieties of PL by HWE and UAE method were both at flowering stage and early fruiting stage,in addition,the range of PLP yield among 24 varieties corresponding to flowering and early fruiting stages were much smaller than other three stages.The optimum picking period of PL are flowering and early fruiting stages.The yield difference of PLP and PSMP by HWE and UAE were compared horizontally and vertically,and the most suitable perilla variety ZB1 was selected.(2)With PL(at flowering and early fruiting stage)and PSM of ZB1 as the research material,PLP and PSMP were extracted by UAE and CEUE method respectively.The extraction process was optimized by RSM,and the optimized process conditions of each extraction method were obtained.The results showed that the yield of CEUE was higher than that of UAE.The main reason for the above results was that the enzymatic hydrolysis biochemical reaction and ultrasonic cavitation effect act synergistically on the raw materials,resulted in more complete destruction of the raw materials from the outside to the inside.The comparative research data also show that UAE has several advantages over CEUE method,such as easier to operate,lesser investment in drugs and equipments,lower energy consumption and easier to expand production.(3)Static adsorption of macroporous adsorption resin,Sevage deproteinized,DEAE-52 cellulose gradient elution and Sephadex G-200 gel chromatography were used to purify PLP and PSMP from ZB1 by UAE.The results of static adsorption test of macroporous resin showed that D101 had the best purification effect.The pigment clearance rates of PLP and PSMP were 60.00±1.30% and 71.37±1.90%.The best deproteinization effect was achieved by repeating 4 times of Sevage reagent treatment,and the protein removal rates of PLP and PSMP were 61.32±2.62% and 65.32±1.87% respectively.Five purified fractions were obtained from PLP and PSMP after gradient elution with different concentrations of Na Cl on DEAE-52 cellulose.Three purified fractions were collected from PLP and PSMP after being purified by Sephadex G-200.After purification,the content of polysaccharides in the purified component of PLP increased from 40.71±1.51% to 76.97±2.35%-89.77±1.52%,as for purified component of PSMP the value increased from 40.17±1.72% to 82.34±1.01%-89.17±1.34%.(4)By comparing the components,micro morphology,monosaccharide composition,physicochemical properties,structure and antioxidant activities of purified ZB1 polysaccharides,the differences between purified components of PLP and PSMP were compared comprehensively for the first time.The research shows that the purification operation did change the moisture content,monosaccharide composition and ratio,thermal stability and microstructure of purified components of PLP and PSMP.PLP and PSMP are mainly composed of rhamnose,ribose,fucose,arabinose,xylose,mannose,glucose and galactose,in addition,PLP and PLP-0.3-I also contain a small amount of glucuronic acid,and PSMP and its purified components contain a small amount of galacturonic acid.The scanning electron microscope(SEM)study shows that microscope morphology of each component were irregular indicating that there is no crystal structure,which is consistent with the XRD results.FTIR analysis showed that each purified component of PLP and PSMP were ?-Glycosidic bond based polysaccharides contained pyranose ring.The thermal stability analysis of PLP and PSMP and their corresponding purified fractions showed that PLP and PSMP had different thermal stabilities,and the impurities in the former were mainly pigments and phenols,while the latter were mainly proteins.The antioxidant study show that the purified fractions of PL and PSMP have well DPPH,OH,ABTS free radical scavenging capacities.and the antioxidative activities of the purified fraction of PLP was better than that of the purified fraction of PSMP.(5)The optimized preparation conditions of perilla leaf purified polysaccharide(PLP-P)effervescent tablets by response surface methodology were as follows: the dosage of PLP-P was 10.08%,the dosage of disintegrating agent was 49.41%,the dosage of sweetener was1.61%,the acid-base ratio was citric acid:sodium bicarbonate=1.2:1,and the dosage of PEG6000 was 3%.It is verified that the PLP-P effervescent tablets prepared under the above conditions can meet the quality inspection requirements,and the comprehensive score was91.16±0.91.The PLP-P effervescent tablets have good antioxidant activities.The ability of PLP-P effervescent tablets to scavenge ABTS free radical is close to that of Vc.