Activity Screening And Structure-activity Relationship Research Of Polysaccharides From Traditional Chinese Medicine Via Immunoaffinity Chromatography

Polysaccharides are a type of natural macromolecular polymers which are composed of aldose and/or ketose linked by glycosidic bonds.They are essential to all living organisms.Glycomics has become the third milestone after the genomics and proteomics in biology.More importantly,it is revealed that polysaccharides have vital bioactivity on regulating immune systems and anti-tumor.However,the current study on the immune activities of polysaccharides is still based on the traditional extraction,isolation,structure analysis,and further immune activity examination via models of pharmacology.This traditional procedure is labor intensive and time-consuming.Macrophages?M??are the main effector cells involved in immunomodulatory by polysaccharides and play key roles in the immune system.The immunomodulatory effects of polysaccharides on M?primarily through excretion of cytokines and regulating the signals to activate the M?.Xanthine oxidase?XOD?is a key enzyme that catalyzes xanthine or hypoxanthine into uric acid.The accumulation of uric acid in the arthrosis leads to its inflammation,which further triggers immune reactions.The oxygen radicals generated during this catalytic process have an impact on the whole immune system.The oxygen radicals are able to regulate immuno activity,synthesis of prostaglandin,and the phagocytosis of the M?.Disruption of the dynamic equilibrium of the oxygen radicals may trigger carcinogenesis,inflammation,pathological damage and autoimmune diseases.It is reported that polysaccharides played their immunomodulatory effects via regulation the activity of XOD.This thesis is aimed at the construction of the Carbon Nanotube Multi-walled modifiedM?lipid rafts immunoaffinity chromatographic system and the XOD enzymatic immunoaffinity chromatographic online system for screening the immune activity of polysaccharides,investigation the immune activity of polysaccharides both in vitro and in vivo,and the establishment of a fast,accurate and durable system for screening the immune activity of the polysaccharides.The main content of this paper includes the following four parts.I Construction of the M?lipid rafts immunoaffinity chromatographic system and the XOD enzymatic immunoaffinity chromatographic online system1)Preparation of the affinity chromatographic stationary phase.First,silica gel-encapsulated multiwalled carbon nanotubes?CNTm@SiO₂?were prepared via the reaction between the acrylated multiwalled carbon nanotubes?CNTm?and the silanized silica gels.Then,human monocytes?THP-1?were induced into M?,which were then cultured and expanded to obtain the lipid rafts from M?by the sucrose gradient high speed centrifugation method.The M?lipid rafts and XOD solution were added into the CNTm@SiO₂ system with continuous stirring in an ice bath to prepare the M?lipid rafts-covered CNTm@SiO₂ chromatographic stationary phase?M?@CNTm@SiO₂?and the CNTm@SiO₂ under the same condition to obtain the XOD-decoratedCNTm@SiO₂chromatographicstationaryphase?XOD@CNTm@SiO₂?.These stationary phases were observed with the scanning electron microscope.The immune activities of M?@CNTm@SiO₂ were examined via the immunofluorescence staining.The activity of the XOD was evaluated via determining the uric acid produced during the catalyzation of xanthine by XOD.2)Investigation of the properties of the affinity chromatographic systems.The two types of stationary phase?M?@CNTm@SiO₂ and XOD@CNTm@SiO₂?were packed into the columns with low pressure.Then the stability,specificity,and the service life of the chromatographic stationary phases?M?@CNTm@SiO₂ and XOD@CNTm@SiO₂?as well as the whole chromatographic systems were evaluated.As a result,the two chromatographic systems had good stability and specificity.The chromatographic columns remained good performance after 240h of continuous use,with longer service life than the traditional lipid rafts chromatographic columns.3)The chromatographic retention behaviors of dextrans with different molecular weights on the two columns were investigated.We found that the Ln value of the molecular weight is inversely proportional to the retention time of chromatographic systems.The good linear relationship of regression equation laid the foundation for subsequent screening and determination of the polysaccharide immune activity.2 Study on online screening of TCM polysaccharide immunoassayFifteen TCM were selected.After the removal of pigments by the organic solvent,the crude polysaccharides were obtained by water extract-alcohol precipitation and deproteinization,followed by further purification using the macroporous resin chromatographic columns,ion exchange chromatographic columns,and gel filtration chromatographic columns.As a result,17 relatively homogenous purified polysaccharide components.The M?@CNTm@SiO2 and XOD@CNTm@SiO2 chromatographic columns were then used to screen polysaccharides with immune activities.1)The separation and purification of polysaccharides:The fifteen medicinal materials were pretreated with petroleum ether and ethanol for decolorizing,which were then extracted with the ultrapure water.The extracts were precipitated with 95%ethanol,and the precipitates were deproteinized by Sevage method.After dialysis for two days against the ultrapure water,the fifteen TCM crude polysaccharides were obtained.The macroporous adsorption resins?such as Ab-8,D-101 and D-315?were adopted for further decolorization.After that,the crude polysaccharides were further purified by ion-exchange chromatography columns and glucan gel columns.2)The identification of the polysaccharide purity:Polysaccharides have no absorbance within the wavelength from 200 nm to 400 nm,and the proteins and nucleic acids exhibit no ultraviolet absorption at 280 nm and 260 nm,respectively.Based on these principles,the UV spectrophotometry was performed to examine the purified polysaccharides.The elemental analyzer was used to detect the elemental content of the purified polysaccharides.The Agilent 1260 high performance liquid chromatography?HPLC?system equipped with the ELSD and 1260-DAD detectors was employed for determination of the purity and molecular weights of the polysaccharides.The results showed that the purified polysaccharides were relatively homogenous with a single molecular weight peak.The purified polysaccharides had high carbohydrate content almost without impurities such as proteins,nucleic acids,small molecules,pigments,and inorganic salts.3)Determination of the polysaccharide content:The phenol-sulfuric acid method was used to detect the total sugar content of polysaccharides.With the concentration of dextran as the horizontal coordinate,the absorbance value was the ordinate.The standard curve was drawn:Y=8.177X-0.0414,R²=0.998,the linear range was good.The total polysaccharide content of these 17 polysaccharides was more than98.5%.4)Determination of protein content:The protein content was detected by the Coomassie brilliant blue method.With the concentration of bovine serum protein as the horizontal coordinate,the absorbance value was the ordinate,and the standard curve was drawn:Y=0.0299X+0.0028,R²=0.9920.The linear relationship was good.The total protein content of these 17 polysaccharides was not more than 0.5%.5)Online screening of the immune active polysaccharides:the 17 purified polysaccharides?2 mg for each?with relatively uniform molecular weights were dissolved in 1 ml of ultrapure water.The polysaccharide solutions were filtrated through 0.45-um membranes,which were then applied to the M?@CNTm@SiO₂ and XOD@CNTm@SiO₂ chromatographic systems for online screening of the immune active polysaccharides.In comparison with the theoretical retention time,the polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1?showed significantly longer retention time on the M?@CNTm@SiO₂ column.Similarly,the retention time of polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1 and 2?were obviously prolonged compared to the theoretical retention time.It was found that the retention time of Flos Lonicerae polysaccharide?fraction 2?and Radix Paeoniae polysaccharide?fraction 1?had the longest retention time,suggesting their strong immune activity.3 In vitro evaluation of polysaccharide immune activity in traditional Chinese medicineIn vitro and in vivo methods were employed to examine the influence of polysaccharides on the activities of XOD and M?and confirm the immune activities of the polysaccharides screened out by the M?@CNTm@SiO₂ and XOD@CNTm@SiO₂ immunoaffinity chromatographic systems.1)The effects of polysaccharides on the phagocytosis of M?in vitro:The UV spectrophotometry was used to investigate the phagocytosis of polysaccharides on M?.The experimental results showed that polysaccharides from Flos Lonicerae?fraction2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1 and 2?could significantly affect M?phagocytosis.2)The effect of polysaccharides on the release of NO and secretion of TNF-?by M?:The ELISA kits were used to detect the contents of NO and TNF-?.The results demonstrated that the polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,Radix Paeoniae?fraction 1 and 2?,Astragalus membranaeus,and Angelica sinensis could remarkably increase the release of NO;meanwhile,Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,Radix Paeoniae?fraction 1 and 2?,and Angelica sinensis significantly enhanced TNF-?secretion ability of M?.3)The effect of polysaccharides on the phagocytosis ability of M?in vivo:The effect of polysaccharide on the phagocytic ability of M?was determined by using the mouse peritoneal M?to phagocytose chicken erythrocytes.It was found that the polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1and 2?significantly increase the phagocytosis ability of M?.4)Inhibition of XOD activity by purified polysaccharides in vitro:The contents of uric acid were detected by ultraviolet spectrophotometry.The reduction of the uric acid content indicated the XOD inhibition activities.It was found that polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1 and 2?could significantly inhibit the activity of XOD.5)Inhibition of XOD activity by polysaccharides in vivo:A high uric acid syndrome rats model was established to detect the XOD activity in serum and liver of rats.The results indicated that polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1 and 2?could significantly decrease the XOD activities in serum and liver.6)Effect of purified polysaccharide on serum uric acid level in rats:The inhibition of XOD activity by polysaccharides could be evaluated by detecting the uric acid concentration.It was revealed that polysaccharides from Flos Lonicerae?fraction 2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,Radix Paeoniae?fraction 1 and 2?,Rhizoma Dioscoreae,Astragalus membranaeus,and Bletilla striata could obviously reduce the blood uric acid in rats.Altogether,these data confirmed the reliability of the M?@CNTm@SiO₂ lipid rafts immunoaffinity chromatography and the XOD@CNTm@SiO2 enzymatic immunoaffinity chromatography systems.The semi-quantitative analytical method established based on the negative control dextrans had a high accuracy.4 The structural analysis of polysaccharides and preliminarily exploration of the structure-activity relationshipThis chapter centered on the preliminary analysis of the structures of the 17purified polysaccharides using the HPLC,infrared spectrum,NMR,periodate oxidation and Congo red test.The primary and advanced structure of polysaccharides was studied,and the structure-activity relationship of the polysaccharides was preliminarily explored in combination with the immune activities of these polysaccharides.1)The relationship between the molecular weight of polysaccharides and the activity:Generally,the polysaccharide with a larger molecular weight was more likely to have more kinds of monosaccharides,glycosidic bonds,and aggregations,leading to a more complex spatial structure which is likely to increase its biological activity.2)The relationship between the monosaccharide composition and the activity:The monosaccharide component of polysaccharide was tested by using the pre-columnar derivatization-HPLC.The polysaccharides which had the main chain structure of glucan and contained arabinose,galactose and mannose showed strong immunological activities.Moreover,the uronic acid could significantly increase the immune activity of polysaccharides.3)The relationship between the type of glycosidic bonds and the activity:The glycosidic bond type of the polysaccharide was analyzed by the high iodate oxidation reaction.The results showed that the polysaccharides from Flos Lonicerae?fraction2?,Achyranthes bidentata,Scutellaria baicalensis,Radix Sophorae,Flammulina velutipes,and Radix Paeoniae?fraction 1 and 2?had a higher proportion of 1?3glycosidic bond than other polysaccharides.The possible reason is that the 1?3glycosidic bond is the non-reductive glycosidic bond with higher stability than the reductive glycosidic bond;therefore,a higher ratio of 1?3 glycosidic bond may lead to a better stability and smaller impact of activity that was caused by the extreme conditions during the extraction and purification of polysaccharides.4)The relationship between the type of the anomeric carbon and the activity:The Fourier transform-infrared spectroscopy and ¹H-NMR analysis were used to analyze the type of the anomeric carbon.The results showed that there was no obvious the correlation between the combination type of the anomeric carbon and the immune activity of polysaccharides.5)The relationship between the advanced structure of the polysaccharide and the activity:The Congo red experiment and transmission electron microscopy were performed to analyze the advanced conformation of polysaccharides.The result of Congo red analysis showed that the polysaccharides that had a triple helix conformation showed stronger immune activity.Transmission electron microscopy analysis confirmed that these polysaccharides exhibited worm chain-like conformation that is similar to the triple helix conformation.