Structural Characterization Of Immunoregulatory Alkali-soluble Polysaccharides From Chrysanthemi Indici Flos

Chrysanthemi Indici Flos, belonging to the Compositae, is a well-known herb with small yellow flowers. It contains many beneficial compounds, including terpenes, flavonoids and volatile oil, polysaccharides, amino acids, proteins and so on. Except for anti-inflammation, analgesic, and antipyretic purposes and the treatment of eye disease in oriental traditional preparations, Chrysanthemi Indici Flos polysaccharide (CIP), is reported to have effects on anti-aging and scavenging reactive oxygen radicals. However, to the best of our knowledge, structure and function are intimately related. In terms of the structure, Jin et al. studied the isolation and structure characterization of a new neutral polysaccharide from Chrysanthemi Indici Flos.The thesis is aimed to study systematically focused on the extraction, purification process of polysaccharide from Chrysanthemi Indici Flos (CIP) including the decolorization, the adsorption of macroresin, removing protein, and column chromatography process. Moreover, the immunostimulation activity of CIP is tested with preliminary analysis its structure. The main contents are as follows:(1) Study on the extraction of crude polysaccharide from Chrysanthemi Indici FlosBy comparing the three methods of extraction (hot water, diluted alkali and enzyme), with the content of crude polysaccharide as index, diluted alkali was choosed as solvent for extraction of crude polysaccharide from Chrysanthemi Indici Flos. On the basis of mono-factor tests, the method of orthogonal test was carried out to determine the optimal condition for the process of the extraction. The optimal process condition using diluted alkali extraction was:the ratio of solid to liquid (1:30), the extraction temperature of60℃, the extraction time of120min, the pH of8and the extraction times of2times. The extraction rate of polysaccharide was40.11%on the above mentioned conditions.(2) Study on the process of decolorizationContrasting three decolorization methods by retained rate of polysaccharide and decoloration rate (hydrogen peroxide oxidation, activated carbon and macroporous resin), it was hydrogen peroxide oxidation process that had the best effect in decolorizing the alkali-soluble olysaccharide from Chrysanthemi Indici Flos. The conditions for decolorization were the time of 4h, the temperature of60℃, the pH of9and the dosage of hydro gen peroxide of8%.(3) Preliminary study in separation and purification by macroporous resinWith three indicators of decoloration rate, deproteinization rate and retained rate of polysaccharide, we studied the purification effect of polysaccharide from Chrysanthemi Indici Flos by static adsorption of six macroporous resin (LSA-700B、LSA-21、D101、XDA-8、 AB-8、XDA-7). LSA-21resin was used to determine the optimum adsorption conditions for this method. The sample was5BV with the current velocity of1.0mL/min meanwhile the sample mass concentration was3.5mg/mL with the pH value of5. The diluted alkali solution (pH8) as eluant was eluted at a flow rate of1.0mL/min.(4) Process of removing proteinBy comparing the three methods of protein removal including by Sevage method, trichloroacetic acid and trypsin-Sevage law with deproteinization rate and retained rate of polysaccharide, trypsin-Sevage method was choosed as the optimal method finally.(5) The immune activity of CIP in miceThe thesis studied the protective effect of CIP on immunosuppressed mice caused by cyclophosphamide. The mice were administered with cyclophosphamide by peritoneal injection to induce an immunosuppressed mouse model. The mice were observed for effect on carbon clearance. The weight of the mice, thymus and spleen were measured; the thymus index and spleen index were calculated.The cell immune function was assessed by delayed type hypersensitivity induced by dinitrofluorobenzene in normal mice.The serum level of hemolysin was determined. All the indices in the model group were reduced (P<0.01).(6) Further purification of CIPOn the basis of the immunological activity, CIP was fractioned by DEAE-52cellulose and Sephadex G-75gel column to afford acidic polysaccharides (CIP-1’, CIP-2’and CIP-3’).(7) Primary structural analysisMolecular weights of the three were8,242,8,383and19,201Da by high performance gel permeation chromatograph, respectively. According to GC, FT-IR and periodate oxidation-Smith degradation, CIP-1’was composed of rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glc) and galactose (Gal) in a molar ratio of5.44:16.40:20.35:3.53:15.43:21.46, with the backbone mostly consisting of (1�'3)-linked-glucopyranosyl and galactopyranosyl residues; CIP-2’ was composed of the same monosaccharides (8.47:13.17:2.43:2.75:14.51:15.76) with the backbone mostly β-(1�'3)-linked-glucopyranosyl and galactopyranosyl; CIP-3’was composed of Rha, Ara, Man, Glc and Gal (17.46:15.88:18.90:63.76:36.44) with the backbone β-(1�'3)-linked-galactopyranosyl residues.