Modification Of Polysaccharides From Cyclocaiya Paliurus And Their Biological Activities

Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) as the sole species in itsgenus belongs to the genus Cyclocarya Iljinskaja (Juglangdaceae), which exhibitsa wide range of biological activities, such as antioxidant, anticoagulant, immunity, etc.As research of glycobiology continues, molecular modification gradually became oneof issues on the study of polysaccharose, for example, sulfation, hydroxymethylation,phosphorylation, etc. Among them, sulfate polysaccharides receive a widespreadattention of research workers because of its excellent antioxidant and antiviral activity.Therefor the aim of the present study is to study the extraction of C. paliuruspolysaccharides, the molecular modification of polysaccharides, and antioxidant ofmodified polysaccharides, which contribute to enlarge applications of C. paliurus.The main studies and results in the paper are mentioned as follows:1. A simple phenol-sulfuric acid spectrophotometry method was developed todetermine the contents of polysaccharides from C. paliurus. The polysaccharides in C.paliurus samples were purified by precipitation with ethanol for removing theinterference components such as lipid, monosaccharide, disaccharide, oligosaccharide,amino acids and protein.followed by extracted with distilled water. The contents ofthe polysaccharides were determined by phenol-sulfuric acid colorimetrictitrationmethod at488nm with glucose as standard. The influence of different time,temperature, different contents of pheno1and sulfuric acid on the contents test ofpolysaccharide was also investigated. Optimum operating conditions were found tobe5%phenol1mL, sulfluric acid4mL, temperature100℃and time20min. At thisoptimum point, the results obtained showed that the method used in this paper wassimple, available, stable and well reproducible, and the average value of the recoveryfor the determination of the polysaccharides was98.2%with2.5%of RSD. Theaverage content of polysaccharide in C. paliurus was4.47%. It indicated that thephenol-sulfuric acid method is suitable for the determination of polysaccharide fromC. paliurus. The concentrations of18kinds of mineral elements (Ca, Al, Mg, K, Fe, Na, Zn, Cr, Mn, Se, Cu, P, Ni, As, Cd, V, Co and Pb) of C. paliurus polysaccharidesamples were determined by inductively coupled plasma atomic emissionspectrometry (ICP-AES). The limits of detection (LOD) of the method for theeighteen elements were in the range of0.01-3.80mg/kg. The average recoveriesobtained by the standard addition method were found between94.34%and105.69%with the RSD in the range of1.01%to4.23%. The results showed that the C. paliuruspolysaccharide samples were abundant in major and trace contents which are healthyfor human body. The average contents of Na, Ca, Al, Mg, K, Fe, P, Zn, Mn, Cr, As,Cu, Pb and Se in polysaccharide samples were137.1±4.2,5980.0±102.7,885.3±25.6,1126.6±42.2,1810.5±30.5,633.0±21.8,2359.1±69.7,31.2±0.3,274.5±10.3,1.7±0.1,7.9±0.1,0.9±0.112.7±0.4, and17.7±0.2mg/kg, respectively.Finally, the levels of elements of Ni, Cd, V and Co were not detected in the samples.ICP-AES is a simple, precise and useful method to determine many mineral elementsin polysaccharide samples simultaneously.2. Polysaccharides from the leaves of C. paliurus were prepared by extractingwith water and removing the interference components such as lipid, protein,monosaccharide disaccharide, oligosaccharide, and amino acids, which is named CP.By chlorosu1fonicacid-pyridine method, the C. paliurus polysaccharide could besulfated successfully. Four sulfated samples of C. paliurus polysaccharide preparedwith CSA–Pyr method were named as S-CP1-1, S-CP1-4, S-CP1-6and S-CP1-8,respectively. A water-soluble polysaccharide extracted from C. paliurus was alsochemically modified to obtain its acetyled derivatives (named as Ac-CP1, Ac-CP2andAc-CP3) and carboxymethylated derivatives (named as CM-CP1, CM-CP2andCM-CP3). Hence, the DS of the chemically modified derivatives was determined. Thephysicochemical properties of its chemically modified derivatives werecomparatively investigated by chemical methods, high-performance gel permeationchromatography, high-performance anion exchange chromatography, fouriertransform infrared spectrometry, circular dichroism (CD) spectra and UV spectramethods. Results revealed that all these modifications were successful. The results ofhigh-performance anion exchange chromatography analysis showed that arabinose, galactose, and glucose were the major monosaccharides constructing the backbonessugar forms in its sulfated derivatives, acetyled derivatives and carboxymethylatedderivatives. and the physicochemical properties of polysaccharides were somechanged after modification. The significant absorption peak of sulfate group could beobserved on FT-IR spectrums. The results of chemical analysis, Fourier-transforminfrared showed that these modifications were successful.3. The antioxidant properties of its sulfated derivatives (named as S-CP1-1,S-CP1-4, S-CP1-6and S-CP1-8), its acetyled derivatives (named as Ac-CP1, Ac-CP2andAc-CP3) and carboxymethylated derivatives (named as CM-CP1, CM-CP2andCM-CP3) were comparatively investigated by DPPH radical assay, hydroxyl radicalassay, superoxide radical assay and β-carotene–linoleic acid assay, respectively.Results showed that the antioxidant properties of polysaccharides were changed aftersulfation. Among these sulfated polysaccharides, the S-CP1-8with relatively lowestdegree of substitution (DS), molecular weight (Mw) and the highest protein contentexhibited excellent antioxidant activities in superoxide, hydroxyl and DPPH radicalassay, and β-carotene–linoleic acid assay. These results indicated that the in vitroantioxidant activities of the sulfated polysaccharides may be related to combinedeffects of sulfate content, DS, Mw, protein and uronic acid content. The antioxidantproperties of the sulfated and unsulfated polysaccharides from C. paliurus in vitrowere also investigated, including hydroxyl, DPPH radicals-scavenging activities. Theresults showed that its sulfated derivatives have prominent antioxidant activity.However, the radical-scavenging activities of all the S-CPs, Ac-CPs, CM-CPs werelower than that of ascorbic acid used in this study. The radical-scavenging activity ofcarboxymethylated derivatives (named as CM-CP1, CM-CP2and CM-CP3) werelower than that of C. paliurus polusaccharide (CP). Introduction of carboxymethylgroups significantly decreased the original polysaccharides’ water solubility so thatthe DPPH radical-scavenging activity decreased, so reduced DPPHradical-scavenging activity and reducing power was found for the polysaccharidessubstituted with carboxymethyl group. The results proved that introducing chemicalgroup can improve antioxidative activity of polysaccharide. Our results also indicated that the introduction of different functional groups and their DS could affect themolecular weight, polarity, solubility, charge density or conformation of nativepolysaccharides, which might lead to the change of antioxidant activities.4. Research results found that polysaccharide isolated from C. paliurussignificantly increased RAW264.7cell proliferation rate and phagocytic activity,which indicated that polysaccharide is low cytotoxic, upregulae the production ofnitric oxide (NO) and cytokines including TNF-α, interleukin (IL)-6, IL-1β. Moreover,the regulation effect on the release of NO, and cytokine secretion was certified to beat transcription and translation level based on the expression of cytokine mRNA,iNOS protein with PCR and Western blot technology. Furthermore, theimmunoregulatory effecs of polysaccharide is related to transcription factor, NF-κBtransactivation and this may be a possible mechanism whereby this herbal medicineelicits its therapeutic effects. The distinct differences between the biologicalresponses of cells treated with C. paliurus polysaccharide compared with thosetreated with aqueous extract are influences on the production of TNF-α andinterleukin (IL)-6. Polysaccharide-treated group augmented the secretion of TNF-α,IL-6, IL-1β dose-dependently while aqueous extract treatd group decreased theproduction of TNF-α, IL-6, IL-1β in a dose-dependent manner in regulatory way,though it uprglated production of TNF-α，IL-6, IL-1β in quantity, besides, the otherimmune enhancement indicators of aqueous extract were much weaker thanpolysaccharide group, which provide further confirmation that C. paliurus couldenhance macrophage function may due to the therapeutic potential of C. paliuruspolysaccharide.