| Pine pollen (Pinus massoniana) is rich in many kinds of nutrition ingredient for humanbody which including nucleic acid, flavonoids and so on. It has anti-aging, regulating immunefunction, anti-fatigue, lowering blood pressure, protecting liver, beauty, weight loss and otherphysiological effects. In this paper, the aqueous extract of pine pollen was taken as raw material,which was separated and purified by HSCCC. The separations were analyzed for purity detectionby HPLC. Hope to get a single material and study the effect of the component on theproliferation and differentiation of3T3-F442A preadipocyte.The main contents are as follows：Firstly, the aqueous extract of pine pollen was preliminary extracted using ethyl acetate,ethanol and chloroform respectively. Then the extracts were used as samples for HSCCCseparation. In order to find the solvent system which had the best separation effect, the extractswere separated in n-hexane-ethyl acetate-ethanol-water system, n-butanol-ethyl acetate-watersystem and chloroform-methanol-water system respectively by HSCCC. The experimentalresults showed that: ethanol extract of the aqueous extract of pine pollen had the best separationeffect in chloroform-methanol-water solvent system (4:3:2). The upper phase was regard asmobile phase, the temperature was25℃, the flow rate was2mL/min, theinstrument host wastransferred, the speed of host was900r/min and the pipe was reversed. The separations werecollected in every two minutes and collected38tubes in all. It`s the first time that the aqueousextract of pine pollen was separated by HSCCC.The separations were detected by HPLC using acetonitrile-water(1:9) as mobile phase. Theresults showed that number14-19tubes had a little impurity. Then they were merged andlabeled component C. The component C was further purified by HPLC in the sameconditions and the collection was labeled component C0. The qualitative analysis ofcomponent C0by HPLC-MS showed that the molecular weight was135.0541. Two stage massspectrometry results showed that the component C0contained ion fragmentations whichmolecular weight was118.0280and91.0188respectively. The chemical formula of component C0wasspeculated to be C4H9NO4or C5H5N5by the Bruker data analysis software.Study on the effect of component C on the proliferation and differentiation of3T3-F442Apreadipocyte. MTT results showed that the experimental groups had a role in promoting cellgrowth compared with the control group. The drug concentration in2μg/mL,4μg/mL,32μg/mLand128μg/mL had significant differences after24h and the drug concentration in4μg/mL,8μg/mL and64μg/mL had significant differences after48h. Oil red O staining showed that theannular lipid droplets appeared in cell differentiation. With the continuous differentiation of cell,cell shape became round or oval, intracellular lipid droplets accumulated and cell volumeincreased. The component C also had a role in promoting cell differentiation compared with thecontrol group. The bigger the drug concentration,the greater the effect. The drug concentration in4μg/mL,16μg/mL,32μg/mL,64μg/mL and128μg/mL had significant differences.Further research on the effect of component C on the proliferation of HepG2humanhepatoma cells. Compared with the blank control group and positive control group, in addition to4μg/mL and32μg/mL (48h), component C had growth inhibition effect on HepG2cellproliferation. Compared with the control group, all drug concentrations had not significantdifferences after24h and48h. It was showed different trends at different time to the sameconcentration. |
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