| Chia Seed is the seed of Euryale Salvia hispanica L,as a "super food",which is widely used in the food,cosmetics and pharmaceutical industries in foreign countries.However,the theoretical research and development and application of Chia seed in China are still in their infancy.The purpose of this paper is to study the polysaccharide and non-carbohydrate compounds of Chia seed.In order to lay a foundation for the further research of Chia Seed and provide theoretical support for the rational development and application of Chia Seed in China.The main contents of this paper are as follows:1.Extraction,purification,structure characterization and antioxidant and immune activities in vitro of Chia Seed Polysaccharide(1)Extraction and impurity removal of Chia seed polysaccharides by water extraction,N-hexane degreasing and acetone deproteinization,and the total polysaccharide was isolated and purified by DEAE Sepharose Fast Flow ion-exchange chromatography column and Sepharose CL-6B gel filtration chromatography column;Two polysaccharides CSP-A and CSP-B,were separated from Chia seed polysaccharide by ion exchange and gel filtration chromatography column.(2)HPGPC-RI method was used to test the purity of purified polysaccharide,and the chemical composition,basic properties and morphology of the pure polysaccharide were analyzed by phenol sulfuric acid method,m-hydroxybenzidine method,bradford method,color difference method,ultraviolet spectrum,infrared spectrum,atomic force microscope,scanning electron microscope,circular dichroism and optical rotation instrument;(3)Structural information of molecular weight,monosaccharide composition and binding sites of pure polysaccharide of chia seed were characterized by HPGPC-MALLS-RI method,PMP pre-column derivatization method,Congo red experiment,oxidation of iodate,Smith degradation,whole methylation experiment and NMR technology;(4)DPPH·and OH·were used to evaluate the antioxidant activity of Chia seed polysaccharide in vitro,and the in vitro immune activity of Chia seed polysaccharide was evaluated by phagocytosis experiment,NO and cytokine TNF-?,IL-6 and IL-1? release experiment;2.Rapid screening and identification of non-carbohydrate compounds and antioxidant activity of chia seeds(1)The characterization and analysis of the non-carbohydrate compounds of Chia seeds based on UPLC-LTQ-Orbitrap-MS technology,and the quantitative analysis of salviaflaside and rosmarinic acid in Chia seeds by HPLC-PDA method;(2)Based on the results of qualitative and quantitative analysis,the total phenol content in Chia seed was determined by forinphenol method,and the antioxidant activity of non-carbohydrate compounds of Chia seed was evaluated by DPPH·.Finally,the possible mechanism of antioxidant activity of Chia seed was further explored by network pharmacology.The main results obtained through the above experiments are as follows:1.CSP-A white,fluffy,It is an acid heteropolysaccharide composed of mannose,glucuronic acid and xylose,and the molar ratio is Man:GlcA:Xyl=l:3:4;The contents of neutral sugar and glyoxylic acid were 35.92%and 60.11%respectively;Molecular weight 1.688 × 105 Da,Polydispersity index Mw/Mn=1.094(±5.074%);The ?=+183°(c=1.0 mg/mL,H2O);Ultraviolet spectra showed strong absorption peak at 200 nm,no absorption peak at 260?280 nm:Infrared spectrum display CSP-A functional groups with a polysaccharide structure and the ? configuration in the CSP-A was detected;AFM results show that CSP-A aggregates and dispersions are irregular in size and shape in aqueous solution;SEM observation CSP-A smooth and smooth surface;CD spectrum shows Cotton effects,explanation that CSP-A exists in asymmetric form in aqueous solution;The Congo red experiment and I2-KI experiment show that there may be three spiral structures and branched chains in the CSP-A;The ?-D-Manp-(1?,?4)-?-D-GlcAp-(1?,?2,4)-?-D-Xylp-(1?,and?4)-?-D-Manp-(l? of glycosidic in the CSP-A were found in combination with oxidation of iodate,Smith degradation,methylation and NMR techniques;2.CSP-B white,fluffy,It is an acid heteropolysaccharide composed of mannose,glucuronic acid and xylose,and the molar ratio is Man:GlcA:Xyl=1:7:9.The contents of neutral sugar,glyoxylic acid and protein were 31.98%,55.41%and 2.3%,respectively;Molecular weight 1.484×105 Da,Polydispersity index Mw/Mn=1.146(±4.849%);The ?=+155°(c=1.0 mg/mL,H2O);Ultraviolet spectra showed the strong absorption peak at 200 nm,no absorption peak at 260-280 nm;Infrared spectrum shows CSP-B functional groups with a polysaccharide structure,and the ? configuration in the CSP-B was detected;AFM results show that CSP-B intermolecular aggregation through cross-linking in aqueous solution,The formation of aggregates and larger aggregates,SEM observation CSP-B smooth and smooth surface;CD spectrum shows Cotton effects,explanation that CSP-B exists in the asymmetric form in aqueous solution;The ?-D-Manp-(1?,?4)-?-D-GlcAp-(1?,?2,4)-?-D-Xylp-(1?,and ?4)-?-D-Manp-(1? of glycosidic in the CSP-B were found in combination with methylation and NMR techniques;3.The results showed that CSP-A and CSP-B had certain scavenging activity on DPPH·and OH·free radicals.In the concentration range of 0.25?4.0 mg/mL,the scavenging activity of Chia seed polysaccharides increased in a concentration dependent manner.The antioxidant activity of the CSP-A was stronger than that of the CSP-B;The phagocytic activity of RAW 264.7 cells was evaluated by the central red method,and it was found that both CSP-A and CSP-B could stimulate RAW 264.7 cells to enhance phagocytic ability(P<0.001).The NO and cytokine release experiments showed that the amount of NO and cytokines released by stimulated RAW 264.7 cells increased in a concentration dependent range of 25?100 ?g/mL(P<0.001).The comprehensive characterization CSP-A and CSP-B both had certain immune promotion effects;4.The results of the rapid screening and identification of non-carbohydrate chemical components of Chia seeds and the research on antioxidant activity are as follows:(1)A total of 48 chemical constituents were identified using UPLC-LTQ-Orbitrap-MS techniques from the Chia seed alcohol extract,including 5 oligosaccharides,5 nucleosides,4 amino acids,30 phenolic acids and 4 other compounds,Among them,phenolic compounds dominate;Establish a HPLC-PDA method for the determination of salviaflaside and rosmarinic acid,The contents of salviaflaside and rosmarinic acid in chia seed were 0.378%and 0.181%,respectively;The determination of total phenol in chia seed by for in phenol method is 1.343± 0.003 mg/g;DPPH·in vitro antioxidant activity experiments showed that the ability to scavenge freedom increased in a concentration dependent range of 0.0625?6.25 mg/mL,and the maximum radical scavenging rate was 91.11%;(2)A network pharmacological study of 23 possible active components of Chia seeds was selected to explore the antioxidant mechanism of Chia seeds,and 68 potential targets of antioxidant stress were obtained.Through protein-protein interaction network analysis,the MAPK1?TNF?PTGS2?AKT1 and CASP3 of these five targets may be the key targets for the antioxidant activity of Chia seeds.KEGG pathway enrichment analysis found that inflammatory response,metabolic abnormalities and inflammatory factor release may lead to oxidative stress,and the molecular mechanism of antioxidant activity of phenolic compounds in Chia seeds is accomplished by regulating multiple targets and pathways.In this study,a preliminary study on the chemical constituents of chia seed polysaccharides and non-carbohydrates was carried out.Among them,the separation,purification,basic structure characterization and pharmacological activities of Chia seed polysaccharides CSP-A and CSP-B were studied;The non-carbohydrate chemical components were analyzed qualitatively and quantitatively,and the network pharmacology method was used to explore the possible mechanism of Chia seeds exerting strong antioxidant activity.In summary,the methods established in this study and the experimental results obtained have certain reference significance for the further research and rational development and application of Chia Seed. |
PDF Full Text Request