Research On Structure,conformation And Rheology Properties Of Water-soluble Polysaccharide From The Seeds Of Cassia Obtusifolia L.

Cassia obtusifolia L.,belonging to Leguminosae family,is an annual herb with great economical and medical importance.The dry and ripe seeds of Cassia obtusifolia L.,semen Cassiae,well known as ‘Jue Ming Zi' in Chinese,were first recorded in ‘Shennong Ben Cao Jing'.The seeds of Cassia obtusifolia have been historically valued as edible and medicinal resources in China,and have been listed in the first batch of ‘Medicinal and Edible Plant' published by Minstry of Health.In China,the seeds of Cassia obtusifolia are widely used as tea,while they are commonly consumed as a substitute for coffee in India.The seeds of Cassia obtusifolia are rich in Ji'an city,which are genuine traditional Chinese medicine in Jiangxi province.The plant's seeds were believed to possess laxative effects and used for the treatment of ophthalmic diseases,headache,hyperlipidemia,and hypertension.Besides,the seeds have been reported to possess liver-protection effects,antibacterial activities,antioxidant properties and neuro-protective effects.A considerable amount of polysaccharides were found in the seeds of Cassia obtusifolia,however,few researches have been reported on the structure and properties of cassia polysaccharide.In this case,the object of the current work was the polysaccharide extracted from the whole seeds of Cassia obtusifolia.Hot water extraction was applied for the isolation of crude polysaccharides(CP)from the whole seeds of Cassia obtusifolia,and two sub-fractions(CP-30 and CP-40)were obtained from CP by gradient ethanol precipitation.The structural characterizations and solution conformation of polysaccharides from the whole seeds of Cassia obtusifolia were analyzed by chemical and instrumental analysis.And the rheological properties of cassia polysaccharides were studied by static and dynamic rheology.The objective of the present work was to provide theoretical basis for the further development and application of cassia polysaccharide.The main results were summarized as follows:(1)Gradient ethanol precipitation was applied to fractionate the water-soluble polysaccharide(CP),and two sub-fractions CP-30(30% ethanol precipitate)and CP-40(40% ethanol precipitate)were obtained.The homogeneity and relative weight-average molecular weight(Mw)of CP and its two sub-fractions were determined by high performance gel permeation chromatography(HPGPC)method.The relative weight-average Mw for CP-30 and CP-40 was estimated to be 9.25 × 105 Da and 0.94 × 105 Da,respectively.Monosaccharide composition and infrared spectra of CP,CP-30 and CP-40 were analyzed.For CP and CP-30,the major monosaccharides were mannose and galactose,while high content of xylose was found in CP-40.The immunomodulatory effects of CP,CP-30 and CP-40 were assessed.All of them were found to possess significant immunomodulation activities,while varied effects of them on macrophage functions were observed,which may due to the differences of their structural characteristics.(2)Methyaltion and gas chromatography-mass spectroscopy(GC-MS)method were applied for the linkage pattern analysis of CP-30,and the structure of CP-30 was further investigated by one-dimensional(1D)and two-dimensional(2D)NMR spectroscopy.All 1H and 13 C NMR were assigned by combining the data from COSY,HSQC,HMBC,and NOESY.The structure of CP-30 was elucidated to be a galactomannan,consisting of ?-1,4-linked-Manp backbone with side chains(T-Galp)attached to O-6 of ?-1,4-linked-Manp.A possible molecular structure of repeating units for CP-30 was proposed as follows:(3)Enzymatic hydrolysis was proposed for the first time to purify the sub-fraction of water-extracted cassia polysaccharide(CP-40),and a homogeneous polysaccharide CP-40-M was obtained with relatively high yield.The structural features of CP-40-M were characterized using high-performance anion-exchange chromatography(HPAEC),methylation and gas chromatography-mass spectroscopy(GC-MS),as well as nuclear magnetic resonance(NMR)spectra.The relative weight-average molecular weight for CP-40-M was determined to be 1.02 × 105 Da,and the molar ratio of xylose to glucuronic acid was 4.62.The structure of CP-40-M was elucidated to be glucuronoxylan,with glucopyranosyluronic acid group terminally attached to O-2 of the ?-1,4-linked-Xylp backbone.It was the first time to obtain this type of xylan from the water extract of Cassia obtusifolia seeds.A possible molecular structure of repeating units for CP-40-M was proposed as follows:(4)The conformational properties of neutral polysaccharide isolated from the whole seeds of Cassia obtusifolia(CP-30)were systematically investigated for the first time,by size exclusion chromatography coupled with multiple detectors(SEC-MALS),dynamic light scattering(DLS),and static light scattering(SLS).DLS and SLS results showed that 0.6 M Na NO3 was a good solvent for CP-30.The structure sensitive parameter ?(1.33-1.80),the Mark-Houwink exponent ?(0.59),and the conformational parameter ?(0.51 from <Rg>z vs Mw,and 0.52 from Rh vs Mw)suggested a random coil conformation of CP-30 in solution.The molar mass per unit of contour length(356.6 nm-1),persistent length(5.3 nm),and characteristic ratio(24.0)obtained by the wormlike cylinder model further indicated a dense random coil conformation of CP-30.By scanning electron microscopy,the linear chain shape of CP-30 in water was observed,which agreed well with the results obtained by light scattering.(5)Static and dynamic rheology techniques were applied for the research on the rheology properties of CP-30.According to the results of static rheology analysis,shear thinning behavior were observed in the solution of CP-30 at high concentration(>1.0%,w/v).The apparent viscosity of the solution was increased with the increase of the addition of sucrose,while temperature,p H value,and addition of lactose and salt had less influence on the apparent viscosity of CP-30.Based on the result of dynamic rheology,CP-30 was not a gelation polysaccharide.Comparisons were made between CP-30 and commercial gums,including chemical compositions,static/dynamic rheology properties,morphology characteristics,and thermal stability.Differences in total sugar content,monosaccharide composition,weight-average molecular weight and intrinsic viscosity were found between CP-30 and commercial cassia gum(CG),which were obtained by different extraction and purification method.The intrinsic viscosity of galactomannans was related to the molecular weight and the mannose to galactose ratio(M/G)of the polysaccharides.The larger the molecular weight,the larger intrinsic viscosity it was.Meanwhile,the higher M/G,the larger intrinsic viscosity it was.By scanning electron microscopy,the linear chain shapes of CP-30 and commercial gums were observed.Both CP-30 and commercial gums possessed similar static/dynamic properties,while different thermal stabilities were found between them.(6)Dynamic rheology was applied for the analysis of synergistic interaction between CP-30 and ?-carrageenan.The synergistic maximum of storage modulus was obtained at 20:80 for CP-30 and ?-carrageenan.Effects of sucrose,lactose,salts,and p H value were evaluated on the viscoelastic properties of CP-30/?-carrageenan mixtures.Sucrose and lactose showed different effects on viscoelastic properties of mixtures,indicating that the viscoelastic property of mixture could be modified by choosing the type and concentration of disaccharides.In a certain concentration range,the storage modulus of the mixture was increased with the increase of the addition of salt.However,at high concentration,the addition of salt decreased the storage modulus of the mixture,which may due to syneresis.With changing p H value in the range of 5.0~8.0,viscoelastic properties of the mixture were weakly influenced.In this case,the p H value of the mixture should be maintained between 5.0 and 8.0.