| In order to enhance the biological activity of polysaccharides, structural modified polysaccharides appeared great significance. There are a lot of modification methods, such as sulfation, carboxymethylation, selenylation, and so on. Sulfated derivation of polysaccharide, which is rich in SO42-, is easier to bind the specific domain of protein. Sulfated modification can change polysaccharides orignal activity.Polysaccharide was extracted and precipitated by 60% alcohol from Pinus massoniana pollen, naming PPM60. Protein was removed by Sevag method. PPM60 was chemically modified by chlorosulfonic acid- pyridine method, naming SPPM60, and its substituted degree was 1.41. There were characteristic absorptions of sulfate ester bond (C-O-S and S=O) showed with infrared ray (IR) spectrum of SPPM60.Five components SPPM60-A,SPPM60-B,SPPM60-C,SPPM60-D,SPPM60-E were obtained separately by molecular sieve Sephacryl S-400HR. Their molecular weight were 1.01×106,6.03×105,1.47×105,3.8×104,1714. We also tested the composition of these five components by High Pressure Liquid Chromatograph (HPLC). SPPM60-A mainly contained glucouronic acid, glucose and galactose; SPPM60-B mainly contained rhamnose, glucouronic acid, glucose and L-arabinose; SPPM60-C mainly contained mannose, glucouronic acid and galactose; SPPM60-D mainly contained mannose, glucose, L-arabinose and an unkown monosaccharide; SPPM60-E mainly contained mannose, rhamnose, galactose, fucose and an unkown monosaccharide.We tested the effects of these five components on the concentration of free calcium in myocardial cells and murine splenocytes. Results showed that SPPM60 could significantly increase the concentration of free calcium ([Ca2+]i) in myocardial cells in vitro, however, PPM60 had inhibition effect. Of the five components, SPPM60- A,B,D increased [Ca2+]i, while SPPM60-C,E reduced. By using the specific L-type calcium channel inhibitor verapamil, we knew that the positive effect of SPPM60-A,B,D may work through L-type calcium channel. Besides, this effect disappeared without extracellular calcium, which could prove that the promotion effect was originated from extracellular calcium.In murine splenocytes, SPPM60 and its five components could also increase [Ca2+]i. The calcium signaling pathway appeared more complex in splenocytes. Verapamil could reduce the promotion effect of SPPM60 and SPPM60-A to E, indicated that they could activate calcium influx through L-type calcium channel, which was as the same as in myocardial cells. Low molecular weight heparin sodium, which is the specific inhibitor of IP3R, could also inhibit their positive effects. The results told us that SPPM60-A to E and SPPM60 could trigger calcium releasing from intracellular stores.
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