| Fortunella margarita (Lour.) Swingle, known as kumquat or cumquat, is a native shrub in south China and is grown for its delicious fruit in many parts of the world, including Europe, Japan, USA, Brazil, Australia, South Africa and India. It is highly nutritious, containing a variety of vitamins, amino acids, non-starch polysaccharides, essential oils, limonoids, flavonoids, etc. In this paper, the effects of preparation methods on the structural properties of polysaccharides from F. margarita (FMPS) were studied. The rheological properties, thermostability and microstruture of FMPS were evaluated. The molecular structural characteristics of FMPS were investigated. The isolation, purification, structural characteristics and in vitro hypolipidemic effect of polysaccharide fractions from F. margarita were studied. Moreover, the hypolipidemic properties and mechanism of FMPS in the hyperlipidemia rats were investigated. The result filled the domestic and international research gap and provided the background and practical knowledge for the comprehensive development and utilization of F. margarita as well as enriched the research connotation of Glycoscience.(1) Structural properties of polysaccharides from F. margarita prepared by different methodsThis chapter used extraction by hot water (WFP), ultrasonic-assisted treatment (UFP), microwave-assisted treatment (MFP) and ultrasonic-microwave synergistic treatment (UMFP), respectively, and then used fourier transform infrared (FT-IR) spectroscopy,1H and 13C nuclear magnetic resonance (NMR) spectroscopy as well as size-exclusion chromatography connected with multi-angle laser light-scattering and refractive index (SEC-MALLS-RI) to characterize the structural properties of polysaccharides. The results showed the optimum conditions of ultrasonic-microwave synergistic method were as follows:ultrasonic power of 131 W, microwave power of 105 W, ratio of water volume to raw material weight of 62 mL/g and extraction time of 120 s. Under these conditions, the yield of polysaccharides could be up to 9.15±0.13%, which was increased by 405.52%、 128.18% and 76.64% compared to WFP, UFP and MFP, respectively. WFP, UFP, MFP and UMFP were all typical carbohydrates according to FT-IR,1H NMR and 13C NMR spectra. The type of glycosidic linkage was mainly the β-glycosidic bond with a small amount of a-glycosidic bond. The results obtained by the SEC-MALLS-RI system showed the molar mass of WFP and UMFP were distributed mainly in the range 5.0×106-1.0×107 g/mol-1 and they accounted for 57.80% and 56.84% of total FMPS, respectively. UFP and MFP were distributed mainly in the range 1.0×106-5.0×106 g/mol-1 and they accounted for 38.24% and 52.39% of FMPS, respectively. The result indicated the ultrasonic-assisted and microwave-assisted extraction methods caused a significant decrease of the molar mass of FMPS but the ultrasonic-microwave synergistic extraction method had no effect on the structural properties of FMPS.(2) Rheological properties of polysaccharides from F. margaritaThe static and dynamic rheological properties of crude F. margarita polysaccharides (C-FMPS) and purified F. margarita polysaccharides (P-FMPS) were studied and compared. The static rheological properties showed that the apparent viscosity of C-FMPS and P-FMPS solution reduced with the saline ionic addition. C-FMPS displayed the great resistant ability on the acid and alkali solution, while P-FMPS had great acid resistant ability with the weak alkali resistant ability. C-FMPS and P-FMPS solution system belonged to the thixotropic system and the shear restoring force of P-FMPS was lower than that of C-FMPS at the same concentration. According to the analysis result of dynamic rheological properties, when the strain was<8.55%, the elasticity modulus (G’) of the C-FMPS solution was always higher than the viscous modulus (G"). When the strain was<2.78%, G’ of P-FMPS solution always exceeded G". When 2% of strain was selected, the system exhibited primarily elastic-dominated or solid-like behavior. In the temperature range of 20-80℃, the dynamic viscosity (η") of the polysaccharide solutions decreased with increasing temperature and G’ value was always higher than G" value, indicating the system exhibited primarily elastic-dominated or solid-like behavior and the polysaccharide network had not been disrupted in this temperature range. Both of solutions were shear-thinning pseudoplastic fluids with flow properties in line with the Power Law model. The apparent viscosity and shear stress increased with the increasing concentration. Moreover, the higher the concentration of polysaccharides, the further the solutions deviated from Newtonian fluids. The pseudoplasticity of P-FMPS was stronger compared to C-FMPS at the same concentration.(3) Thermostability and microstructure of polysaccharides from F. margaritaThe thermostability of C-FMPS and P-FMPS was studied using differential scanning calorimetry (DSC) and dynamic mechanical analyzer (DMA). The molecular weight and microstructure were investigated by SEC-MALLS-RI, scanning electron microscope (SEM) and confocal scanning laser microscope (CSLM), respectively. The result showed the oxidative decomposition temperature of P-FMPS was higher than that of C-FMPS and the greater energy was released from P-FMPS in the process of oxidation decomposition. In the scanning temperature range of 25-200℃, G’ and G" values of P-FMPS were greater than those of C-FMPS. G’ of P-FMPS changed gently while G’of C-FMPS changed steeply. The peak of G" of P-FMPS was wide and flat while C-FMPS was narrow and high. P-FMPS was more stable than C-FMPS under the condition of high temperature. The result by SEC-MALLS-RI showed the molecular weight distribution and molecular weight of P-FMPS were greater than those of C-FMPS. The surface of C-FMPS was rough and dense whereas P-FMPS displayed a smooth network structure by SEM. According to confocal laser scanning microscopy, C-FMPS dispersed in the medium without connected network, whereas the network of P-FMPS was unevenly distributed in the medium and the shape was compact and smooth. The molecular weight and microstructure of P-FMPS were attributed to the purification process while rearrangement and aggregation of polysaccharide molecules took place, which resulted in the significant difference of rheological properties and thermostability between C-FMPS and P-FMPS.(4) Molecular structural characteristics of polysaccharides from F. margaritaThe molar weight (Mw), polydispersity index (Mw/Mn), root-mean-square (RMS) turning radius (Rw), molar mass distribution, radius distribution and chain conformation of FMPS were investigated systematically by SEC-MALLS-RI. The Mw, Mw/Mn and Rw values were 6.192×106 (±0.814%) g/mol,17.376 (±7.974%) and 42.1 (±9.2%) nm, respectively, indicating FMPS was a highly dispersed macromolecule polymer. According to the result of molecular weight distribution, the molar mass of FMPS was distributed mainly in the range 1×105-5×106 g/mol and it accounted for 86.48% of total polysaccharides. According to the result of polysaccharide molecular peak distribution, the molecules of four peaks (P1, P2, P3 and P4) accounted for 19.00 (±1.13%),31.60 (±2.12%),31.00 (±2.85%) and 18.40 (±1.74%) of the total molecules of FMPS, respectively. P2 and P3 as the predominant constituent, accounted for 62.60% of the total polysaccharides suggesting that the molecular weight of FMPS was mainly distributed in the ranges of 1.828×105-9.563×105 g/mol. FMPS was composed of four polysaccharide fractions with different molecular weight. According to the result of molecular radius distribution, the radius of FMPS was distributed mainly in the range 20-50nm and it accounted for 88.79% of total polysaccharides. The molecular weight and radius of FMPS did not display positive correlation. FMPS displayed an uniform spherical conformation in solution. These results indicated FMPS was a kind of complex heteropolysaccharides with different molecular fragments. It will provide the theoretical basis for the further investigation of the isolation, purification, structure-activity relationship and active mechanism of FMPS.(5) Isolation and purification of polysaccharide fraction from F. margaritaFMPS was isolated and purified by the linear and gradient elution methods of DEAE Sepharose CL-6B column, respectively. Each of polysaccharide fragments was further purified by Sephadex G-100 column. The purity of polysaccharide fraction was identified by high performance liquid chromatography (HPLC) and Uv-vis spectrum scanning method, separately. The result showed polysaccharide fragments were not isolated completely by linear elution method whereas polysaccharide fragments were obtained completely by gradient elution method. After the purification by Sephadex G-100 column, four polysaccharide fractions (FMPS1, FMPS2, FMPS3 and FMPS4) with different molecular weight were obtained, which was consistent with the result of molecular structural characteristics of FMPS. The result of purity identification by HPLC indicated FMPS1, FMPS2, FMPS3 and FMPS4 were homogeneous polysaccharide components with relatively narrow molecular weight distribution. The results by UV-vis spectroscopy showed no peaks at 260-280 nm, indicating there was no protein and amino acid in the four polysaccharide fractions.(6) Structural characteristics of polysaccharide fractions from F. margaritaThe structural properties of four polysaccharide fractions (FMPS1, FMPS2, FMPS3 and FMPS4) were characterized by FT-IR, SEC-MALLS-RI,1D NMR and 2D NMR. The result showed FMPS 1 and FMPS3 were mainly pectic polysaccharides. FMPS1 and FMPS2 were mainly linked by a-glycosidic linkages with relative small β-glycosidic linkages, FMPS3 were mainly linked by β-glycosidic linkages with relative small a-glycosidic linkages while FMPS4 only linked by β-glycosidic linkages. Mw values of FMPS1, FMPS2, FMPS3 and FMPS4 were 2.572x107 (±0.517%),1.755×106 (±2.009%),2.563×105 (±1.784%) and 2.411×105 (±1.808%) g/mol, respectively. Mw/Mn values were 1.090 (±0.708%),1.220 (±2.813%),1.124 (±2.508%) and 1.842 (±2.509%), respectively. Rw values were 44.6 (±0.8%),33.4 (±6.7%),15.3 (±27.5%) and 9.4 (±69.6%) nm, respectively. In aqueous solution, FMPS1 existed as a globular shape conformation; FMPS2 existed as a random coil conformation while FMPS3 and FMPS4 were highly branched polymers. According to the result by 1H-NMR、13C-NMR、COSY、HMBC、HSQC、NOESY and TOCSY 2D NMR, FMPS1 was mainly composed of α-Glc、α-GalA、β-Rha and β-Galp and its preliminary structural unit was α-Glc-β-Galp-β-Rha-α-GalA-α-Glc-. FMPS4 was mainly composed of α-Ara、α-Glc、β-Rha、β-Galp and β-Man and its preliminary structural unit was-α-Ara-β-Rha-β-Galp-α-Glc-β-Man-α-Ara-.(7) Hypolipidemic effect in vitro and structure-activity relationship of polysaccharide fractions from F. margaritaThe pancreatic lipase activity inhibition, bile acid-binding activity and antioxidant activity in vitro of polysaccharide fractions from F. margarita were studied. The relationship between structure and activity of FMPS1, FMPS2, FMPS3 and FMPS4 was investigated and compared. Among the four polysaccharide fractions, FMPS1 and FMPS3 displayed the stronger inhibitory effect on the pancreatic lipase, which was affected by their monosaccharide composition and molecular weight; FMPS1 and FMPS2 displayed the stronger ability to bind bile acids, which was affected by their molecular weight; and FMPS3 and FMPS4 displayed the greater antioxidant activity, which was by their monosaccharide composition, category of glycosidic bond, molecular weight, molecular radius and chain conformation in solution. The hypolipidemic effect of polysaccharide fractions from F. margarita was a result of a combination of multiple lipid metabolic pathways. Interestingly, the hypolipidemic property of polysaccharide fractions from F. margarita was a result of a combination of multiple structural factors rather than a single factor.(8) Hypolipidemic mechanism of polysaccharides from F. margarita in the hyperlipidemia ratsThis study used rat as the research object. The fat metabolic disorder model of rat was formed by feeding high-fat diet. The rats fed normal diet were used as a normal control group and the rats fed simvastatin with high-fat diet were used as a positive control group. The effect of low dose, medium dose and high dose FMPS on the serum lipid level and antioxidant index of plasma and tissues in the hyperlipidemia rats. The result showed the contents of TQ TC, LDL-C and NEFA reduced and HDL-C and LIPA increased significantly by feeding FMPS in the hyperlipidemia rats. Meanwhile, the abilities of SOD、GSH-Px, GST and T-AOC enhanced and the content of MDA reduced by feeding FMPS in the hyperlipidemia rats. There was a certain concentration-response relationship. Moreover, the body weight, liver and spleen index of the hyperlipidemia rats reduced significantly, which was relative to the concentration of FMPS. Histopathological micrographs of hepatic tissue and blood vessel morphology of the hyperlipidemia rats showed the fat deposition in liver cells reduced and the vascular endothelial cells were protected by feeding FMPS in the hyperlipidemia rats. The result indicated FMPS displayed significant regulatory role on the lipid metabolism disorder of the hyperlipidemia rats. Combination with the hypilipidemic effect of polysaccharide fractions in vitro, the hypolipidemic mechanism of polysaccharides from F margarita in the hyperlipidemia rats was achieved by increasing the lipase activity, reducing the content of lipid and enhancing the activity of antioxidant enzymes, etc. |
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