The Structure Determination Of Pinus Massoniana Pollen And Effect Of Sulfated Polysaccharide On The Proliferation Of HepG2

It had been showed by multiple studies that polysaccharide,as a kind of important bioactive macromolecule,has a variety of functional activities,e.g.it features oxidation resistance and anti-tumor activity and can regulate blood sugar level and improve immunity of the organism,etc.By particular modification of polysaccharide,the biological activity of polysaccharide could be significantly improved.It had been proved by the early-stage work of the laboratory that sulfated polysaccharide of pinus massoniana pollen SPPM60 could inhibit the proliferation of hepatoma cell Hep G2 notably,while un-sulfated polysaccharide PPM60 had no significant influence on the proliferation of Hep G2.In addition,SPPM60 could reduce the intracellular calcium ion concentration of Hep G2,while PPM60 had no significant influence on [Ca²⁺]i.A large number of experiments showed that the biological activity of polysaccharide could be improved through degradation of it with proper methods.At present,there were few researches on the degradation of polysaccharide of pinus massoniana pollen and the structure-function relationship of polysaccharide after degradation.Therefore,on the basis of combination of the early-stage work of the laboratory,degradation of polysaccharide components of pinus massoniana pollen was carried out,and the difference between the structure-function relationship before and after degradation was explored through cellular experiment and NMR technology.The experiment mainly included the following parts:I.Polysaccharide was extracted from wall-disrupted pinus massoniana pollen with the method of water boiling and precipitation with ethanol,impure protein was removed with trichloroacetic acid method,and the polysaccharide precipitated with 80% ethanol was separated and purified with TOYOPEARL HW-65 S medium pressure sephadex column;relatively uniform polysaccharide components were got after multiple repetitions,which were named PPM80-B.The relative molecular mass of polysaccharide components PPM80-B was determined with high performance gel chromatography,and the determination result was 317.8K.II.Acidolysis method was adopted to degrade polysaccharide of pinus massoniana pollen PPM80-B,and the polysaccharide components with low molecular mass generated after the degradation were got,named DPPM80-B.The relative molecular mass of DPPM80-B was determined under the same chromatographic conditions with those in the first experiment,and the result was 3.131 K,indicating a successful degradation.Cholorsulfonic acid-pyridine method was applied to sulfate and modify PPM80-B and DPPM80-B respectively to get sulfated polysaccharide,named SPPM80-B and DSPPM80-B respectively.Barium chloride-gelatin turbidimetry method was used to determine the sulfate substitution degree of SPPM80-B and DSPPM80-B respectively,and the results for the two were 1.08 and 1.26 respectively.III.The influence on the proliferation of hepatoma cell Hep G2 of four kinds of polysaccharide?PPM80,SPPM80,DPPM80 and DSPPM80?was explored with MTT method respectively,and the experiment results showed that SPPM80 and DSPPM80 had inhibiting effect on the proliferation of Hep G2,with time and dosage dependence.The inhibiting effect of SPPM80 on the proliferation of Hep G2 was stronger than that of DSPPM80.The inhibiting rate of 200?g/m L SPPM80 on Hep G2 after 72 h could reach 31.47%,while under the same conditions,that of DSPPM80 was 15.38%.PPM80 and DPPM80 had no significant influence on the proliferation of Hep G2.IV.The influence on the intracellular calcium ion concentration of hepatoma cell Hep G2 of four kinds of polysaccharide?PPM80,SPPM80,DPPM80 and DSPPM80?was determined with fura-2 probe and dual-wavelength fluorescent technique,and the results showed that SPPM80 and DSPPM80 could reduce the intracellular calcium ion concentration of hepatoma cell Hep G2,and SPPM80 had more significant effect.PPM80 and DPPM80 had no significant influence on the intracellular calcium ion concentration of Hep G2.V.The structure of pine pollen polysaccharide was analyzed by nuclear magnetic resonance spectroscopy,mainly from ¹HNMR and 13 CNMR.The results show that the monosaccharide constituting of polysaccharide is present in the form of a pyran ring and the polysaccharide is a neutral polysaccharide which does not contain a Gal N residue.This polysaccharide may contain methyl hydrogen in an acetyl group or in N-acetylamino group with the presence of C-6 deoxyglycosylmethyl.In conclusion,results of the experiment showed that sulfated polysaccharide of pinus massoniana pollen could inhibit the proliferation of hepatoma cell Hep G2 and the inhibiting function on the proliferation of Hep G2 of undegraded polysaccharide components were stronger than that of degraded ones.In addition,sulfated polysaccharide of pinus massoniana pollen could reduce the intracellular calcium ion concentration of Hep G2 notably,which might also be a method for sulfated polysaccharide of pinus massoniana pollen to regulate the cellular activity of Hep G2.