Studies On The Structure,immunomodulatory And Anti-inflammatory Activities Of Erythronium Sibiricum Bulb Polysaccharides And Erythronium Sibiricum Bulb's Quality Standard

Erythronium sibiricum bulb has been used as a traditional herbal medicine for centuries by the Kazak ethnic population in Xinjiang.However,little is known about the bioactive components in the bulb.The effect of this medicinal herb which can relieve waist and knee soreness and improve physical symptoms of weakness is consistent with the immunomodulatory of polysaccharide.So the present study aimed to extract,purify the crude polysaccharides,and then explore structure characterisation and biological activity about purified polysaccharides.On the other hand,the establishment of quality standards for Erythronium sibiricum bulb is the second purpose of this paper.Objective:(1)Study on extraction,purification,chemical properties and structural characterization of ESBP.(2)To observe immunomodulatory and anti-inflammatory effects of ESBP and its homogenous polysaccharides.(3)To establish quality standards for Erythronium sibiricum bulb and write draft quality standards and drafting instructions.Methods:(1)In this study,the extraction of Erythronium sibiricum bulb polysaccharides(ESBP)through hot water decoction was optimised using response surface methodology(RSM)and a three-level,four-factor Box-Behnken design,and then ESBP was purified through an ion-exchange chromatography of DEAE Sepharose CL-6B and gel-filtration chromatography of Sephadex G-100.The homogeneity and molecular weight of homogenous polysaccharides were identified by HPGPC.(2)Total sugar content was determined using the phenol-sulphuric acid method.Uronic acid content was estimated using the m-hydroxydiphenyl method.Iodine-potassium iodide reagent was added to the polysaccharide solution to detect the presence of starch.Pre-colum derivatization-HPLC method was used to analyse monosaccharide composition.Infrared spectrum,Smith degradation,methylation analysis,partial hydrolysis experiment and NMR studies were used to reveal the structural characterization of homogenous polysaccharides.(3)Non-specific immune regulation function of ESBP was studied by carbon particle clearance test in mice,and the anti-inflammatory and analgesic activities of ESBP in mice were investigated using xylene-induced ear oedema test,cotton pellet-induced granuloma test and acetic acid-induced writhing response test.The murine macrophage cell line RAW264.7 was used to observe immunomodulatory effects of ESBP and its homogenous polysaccharides,CCK-8 method was used to detect cell proliferation,then neutral red method,Griess reagent and ELISA were used to determine the effects on the phagocytosis,NO release and cytokine production of RAW264.7 cells.The influences of ESBP and its homogenous polysaccharides on inflammatory response induced by LPS in RAW264.7 macrophages were investigated to identify inflammatory effects.(4)According to "Chinese Pharmacopoeia" technical requirements of TCM quality standard,moisture and ash of ten batches of Erythronium sibiricum bulb samples were analysized.Microwave digestion and ICP-MS method was used to determine the contents of elements and heavy metal elements in Erythronium sibiricum bulb samples;Pesticide residues contents of samples were analyzed by acetonitrile ultrasonic extraction and GC-MS method;TLC Methods for the identification of amino acid and polysaccharides were established,respectively.And pre-colum deribatization-HPLC method for the determination of polysaccharide content was also completed.Results:(1)The optimal experiment yield of 37.25%± 0.17% was obtained under the following optimum conditions: extraction time of 4.3 h,extraction temperature of 90?,ratio of liquid to raw material of 37 ml/g and extraction cycle number of three.The aforementioned experimental yield of ESBP closely agreed with the predicted value(37.465%).Four homogeneous and water-soluble polysaccharides(ESBP1-1,ESBP1-2,ESBP2-1,ESBP3-1)with molecular weights of 1.3×104?1.7×104?9.4×105?4.1×105 Da were extracted from ESBP,which was purified through an ion-exchange chromatography of DEAE Sepharose CL-6B and gel-filtration chromatography of Sephadex G-100.(2)The total sugars of ESBP1-1,ESBP1-2,ESBP2-1,ESBP3-1 were 97.8%,98.7%,99.3%,97.0%,respectively.The uronic acid contents of ESBP2-1 and ESBP3-1 were 0.02% and 1.9%,but there is no uronic acid in ESBP1-1 and ESBP1-2.The starch contents of ESBP1-1 and ESBP1-2 were 0.5% and 0.3%,but there is no starch in ESBP2-1 and ESBP3-1.The monosaccharide composition results showed that ESBP1-1 and ESBP1-2 were composed of glucose totally.ESBP2-1 is composed of glucose,galactose and arabinose in a ratio of 24.3: 1.1: 1 and ESBP3-1 is composed of glucose and galactosen in a ratio of 14.8:1.Four monogeneous polysaccharides have a same structural characteristic,which the repeating units is a ?1)-?-D-Glcp-(4? backbone where the branching occurs at the O-6 positions of the residues,but the branched stucture of them is different.(3)The results of carbon particle clearance test showed that ESBP had the abilities to improve the organ index and clearance index in immunosuppressed mice.Cylene-induced ear oedema test,cotton pellet-induced granuloma and acetic acid-induced writhing response test in mice revealed that ESBP had the abilities to inhibit the ear swelling,reduce the quality of granulation and reduce the number of writhing.ESBP and homogenous polysaccharides could significantly increase cell viability,improve phagocytosis,and incresde the secretion of NO and the production of cytokines TNF-? and IL-1? on RAW264.7 cells.ESBP and its homogenous polysaccharides can decrease NO,TNF-? and IL-1? production on inflammatory response induced by LPS in RAW264.7 macrophages.(4)Quality control methods for Erythronium sibiricum bulb were established,and the study included identification(traits,Microscopic identification and TLC),check(moisture,ash,heavy metals and pesticide residues),and content determination(HPLC).The quality standard draft was developed.Conclusion:(1)Extraction process and regression model in this study were accurate and adequate for ESBP extraction.(2)The purification methods using an ion-exchange chromatography of DEAE Sepharose CL-6B and gel-filtration chromatography of Sephadex G-100 were feasible for ESBP.Four homogeneous polysaccharides were obtained,all of them contained a large amounts of ?1)-?-D-Glcp-(4? in the backbone.(3)Polysaccharides of Erythronium sibiricum bulb have the effect of enhancing immunity,may be related to polysaccharides that can enhance cell viability,increase phagocytosis and the expression of cytokines.On the other hand,polysaccharides of Erythronium sibiricum bulb have anti-inflammatory activity,may be associated with polysaccharides that can inhibit the inflammatory factors caused by the excessive immunity.These results demonstrate that Polysaccharides of Erythronium sibiricum bulb regulate immune reaction in a bi-directional way.(4)The draft of quality standards and drafting instructions were developed.Through the methodological investigation and system verification,it showed that the methods were simple,stable and feasible.