Isolation, Purification, Structure Determination And Biological Activities Of Polysaccharides From Bulb Of Lily (Lilium Lancifolium Thunb.)

Recently, lily polysaccharides(LLPS) have attracted a great deal of attention for their effect on antioxidant, immunomodulatory, anti-tumor and hypoglycemic. At present, researches on LLPS are mostly focused on the crude polysaccharide. However, there are still lack of reports concerning the isolation, purification, structure and biological activities of LLPS.Tiger lily, Lilium lancifolium Thunb., is the highest medicinal value in the three edible lily varieties in China. In this dissertation, the isolation, purification, basic physical and chemical properties, chemical structure and antioxidant and immunomodulating activity in vitro of polysaccharides from Tiger lily were investigated. The main results were concluded as follows:1, Crude LLPS was extracted by using methods of hot water assisted with ultrasonic wave and extraction yield was 9.46%. The crude LLPS was separated firstly through DEAE-Sepharose Fast Flow chromatography column and further purified through chromatography column of Sephadex G-100. Three fractions obtained were named as LLPS-1, LLPS-2 and LLPS-3 and the yield of them were 1.66%,1.71% and 0.28%, respectively. The purity of LLPS-1, LLPS-2 and LLPS-3 was confirmed by using high performance liquid gel filtration chromatography and results showed that LLPS-1, LLPS-2 and LLPS-3 were homogeneous polysaccharides. Their average molecular weights were 350.51 kDa,403.29kDa and 146.18 kDa, respectively.2, The contents of total carbohydrates, protein, uronic acid and sulfuric radical in crude LLPS, LLPS-1, LLPS-2 and LLPS-3 were determined according to the reported methods of phenol-sulfuric acid coloration, coomassie brilliant blue coloration, sulfuric acid-carbazole coloration and barium chloride-gelatin coloration, respectively. The contents of total carbohydrate in the four polysaccharides were 78.01%,97.40%,96.19% and 79.69%, respectively; protein contents of crude LLPS were 3.61%, but no protein in LLPS-1, LLPS-2 and LLPS-3 was cheched out; uronic acid contents were 6.33%,14.83%, 12.19%and 37.34%, respectively; sulfuric radical contents were 1.34%,0.55%,0.43% and 0.65%, respectively, no starch and phenols in LLPS-1, LLPS 2 and LLPS-3 was found.3, Structure determination of LLPS(1) The results of elemental analysis showed that LLPS-1, LLPS-2 and LLPS-3 were mainly composed of C and H elements, contained traces of S element, without N element.(2) Monosaccharide composition analysis was carried out by high performance anion exchange chromatography. LLPS-1 backbone chain was mainly composed of glucose and mannose in a molar ratio of about 2:1, containing traces of arabinose (there was an arabinose at interval of about 42 sugar units), might contain a very small amount of fucose andgalactose. LLPS-2 backbone chain was mainly composed of glucose and mannose in a 1:1 molar ratio, might contain trace amounts of arabinose (there was an arabinose at interval of about 53 sugar units). LLPS-3 backbone chain was mainly composed of arabinose, galactose, glucose and mannose in a molar ratio of about 2:2:2:1, contained trace amounts of galacturonic acid (there was a galacturonic acid at intervalof about 34 sugar units).(3) Spectra of FTIR and NMR both implied that there were pyranose rings in LLPS-1, LLPS-2 and LLPS-3. LLPS-1 and LLPS-2 showed feature of the β-glycosidic bond, LLPS-3 exhibited character of both α- and β-glycosidic bond. Spectra of FTIR also showed that β-D-glucose and β-D-mannose existed in LLPS-1 and LLPS-2, a-D-mannose, a-D-galactose and rhamnose existed in LLPS-3.(4) Results of periodate oxidation, Smith degradation suggested that the backbone chain of LLPS-1, LLPS-2 were linked mainly by (1,4)-glycosidic bond, with a small amount of (1,2)- and (1,6)- glycosidic bond. Glucose and mannose residue also contained (1,3)-glycosidic bond. LLPS-3, mannose, arabinose and galactose residue mainly contained (1,3)-glycosidic bond, also contains (1.4)-, (1,2)- and (1,6)- glycosidic bond.(5) X-ray diffraction analysis indicated that LLPS-1, LLPS-2 and LLPS-3 showed the amorphous state, with little crystallization of the polysaccharide or polysaccharide complexes. Degree of crystallization was very low, and contained only a trace amount of crystal structure.4, Antioxidant activity in vitro of LLPS was evaluated in different system. Results indicated that the crude LLPS, LLPS- 1 and LLPS-2 had a strong scavenging capacity of OH-radical, moderate scavenging ability of H2O2, and weak reducing power and scavenging activity of O2· radical. Within a certain range of concentrations, the antioxidant activity was enhanced with the increasing concentration. Overall, the order of the antioxidant activity was crude LLPS> LLPS-2> LLPS-1.5, Immunomodulating activities in vitro of LLPS were measured by using macrophage RAW 264.7 model experiments. Results showed that, proliferation of macrophage increased and then decreased with the increasing concentration (25-400 μg/mL) of LLPS-1 and LLPS-3, and reached their maximum at the concentration of 100 μg/mL. The effect of LLPS-2 on proliferation of macrophage had been increasing, and there was no significant difference compared with positive control LPS at the concentration of 400 μg/ mL. The ability of LLPS to stimulate macrophages to devour neutral red was enhanced at first and then slightly weakened with the increasing concentration. At the range concentration of 50-400 μg/mL, LLPS were extremely significantly higher than that of the blank control (p< 0.01). LLPS could promote NO production of macrophages and their activities were dose-dependent. They would be able to significantly promote macrophages to release NO at low concentration (25 μg/mL) and NO production was extremely significantly higher than that of the blank control and positive control at the concentration of 400 μg/mL (p< 0.01). In general, the order of immunomodulating activity in vitro was LLPS-2> LLPS-3> LLPS-1.