| Pinus massoniana Pollen has a high content of protein, amino acid, polysaccharide, vitamin and microelement, which can stimulate the immune function, and show anti-tumor, anti-aging, and other effects.The studies were conducted on the extraction, purification, physicochemical properties, chemical modifications, structural analysis and bioactivity of Pinus massoniana Pollen Polysaccharides. The main results are as follows:1. Isolation and Purification of Pinus massoniana Pollen polysaccharidesPolysaccharide was extracted and precipitated by 60% alcohol from Pinus massoniana pollen, naming PPM60. Fore components PPM60-A, PPM60-B, PPM60-C, PPM60-D were obtained separately by molecular sieve Sephacryl S-400HR. Their molecular weight were 1.4×106, 1.2×106, 8.6×104, 5.7×104.2. Chemically modified by chlorosulfonic acid-pyridine method of Pinus massoniana Pollen polysaccharidesPPM60 and its five components were chemically modified by chlorosulfonic acid-pyridine method, naming SPPM60, SPPM60-A, SPPM60-B, SPPM60-C, SPPM60-D and their substituted degree were 1.44, 1.40, 1.32, 1.57.3. Monosaccharide composition of polysaccharides and sulfonic esterificated polysaccharidesThe analysis of monosaccharide composition in the polysaccharide by HPLC revealed that PPM60-A was composed of mannose, an unknown monosaccharide, galactose and fucose in the molar ratios of 5.76:2.73:3.35:1; SPPM60-A was composed of mannose, an unknown monosaccharide, galactose and fucose in the molar ratios of 6.38:3.91:5.21:1; PPM60-B was composed of mannose, an unknown monosaccharide, galactose and fucose in the molar ratios of 1.45:0.82:1.54:1; SPPM60-B was composed of mannose, an unknown monosaccharide, galactose and fucose in the molar ratios of 2.37:0.68:0.75:1; PPM60-C was composed of mannose, an unknown monosaccharide, galactose and fucose in the molar ratios of 9.50:3.55:4.76:1;SPPM60-C was composed of mannose, an unknown monosaccharide, galactose and fucose in the molar ratios of 2.85:0.98:1.11:1; PPM60-D was composed of mannose, glucose, an unknown monosaccharide and galactose in the molar ratios of 1.95:0.66:0.41:1; SPPM60-D was composed of mannose, glucose, an unknown monosaccharide and galactose in the molar ratios of 2.31:0.55:0.55:1.4. The effects of polysaccharides and sulfonic esterificated polysaccharides on the concentration of free calcium in myocardial cellsThe concentration of intracellular free calcium in myocardial cells was measured by Fura-2 /AM. PPM60, PPM60-A, PPM60-B, PPM60-C, PPM60-D, SPPM60, SPPM60-A, SPPM60-B, SPPM60-C and SPPM60 -D could significantly increase the concentration of free calcium ([Ca2+]i) in myocardial cells in vitro, while the sulfonic esterificated polysaccharides have more significant effect of increasing the concentration of free calcium ([Ca2+]i) in myocardial cells.5. Structural analysis of Pinus massoniana Pollen polysaccharidesWe analysise the structure of PPM60-A and SPPM60-A through ultraviolet (UV) spectrum infrared ray (IR) spectrum, nuclear magnetic resonance (1HNMR,13CNMR), atomic force microscopy (AFM).The main results are as follows:The ulrtaviolet spectrum showed that PPM60-A and SPPM60-A had not absorption in 260nm and 280nm, and it accounts for no proteins and nuclear acids in PPM60-A and SPPM60-A.It was indicated by IR that PPM60-A was neutral polysaccharides which was mainly composed of pyranose.Sulfonic esterificated PPM60-A was synthesized by Pyridine-sulfonic acid method. The characteristic absorbance of the sulfonic esters was determined by IR.IH-NMR showed that the configuration of PPM60-A and SPPM60-A are a-glycosidic andβ-glycosidic. PPM60-A and SPPM60-A have characteristic absorption peaks of fucose methyl.According to 13C-NMR, PPM60-A and SPPM60-A contain four monosaccharide residues, and was mainly composed of pyranose.Through AFM observations of PPM60-A and SPPM60-A, it was found that PPM60-A aggregated into well-Proportioned, little round particles, with diameter about 20-40nm and height about 1-3nm. SPPM60-A has blame-like protuberance, and average height of SPPM60-A was about 20-70nm, average diameter of SPPM60-A was about 5-8nm.Periodate oxidation and Smith degradation proves that the chain of PPM60-A consisits (1→6) and (1→2) glycosidic bond,and chain of SPPM60-A consisits (1→6) glycosidic bond.
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