| Helianthus annuus L.,also known as sunflower.According to the Pharmacopoeia,many parts of sunflower have been recognized with high medicinal value.Water soluble constituents of the wick of sunflower stem may indeed possess anticancer activity.In an effort to search for anti-tumor substances from the wick of sunflower stem,polysaccharide has been isolated and identified by our group.So polysaccharide isolated from the wick of sunflower stem was first time studied on inhibiting tumor metastasis mechanism,and the structure was discussed preliminarily.The main results are as follows.1.The water extraction and alcohol precipitation method was used to extract the wick of sunflower with the polysaccharide yield as index.In this study,the optimal conditions were obtained.These data were as follows:extraction time was twice,mesh number was 60~80,solid-liquid ratio(g/m L)was 1:50,extract time was 3.0 h,and extract temperature was 90~oC.The maximum extraction rate(6.56%)was achieved under the optimal extraction conditions.The maximum extraction yields of polysaccharides were up to 266.03 mg/g under the optimal extraction conditions.2.The extracted polysaccharides were processed by removing protein,removing pigment.Four main components were isolated and purified from the extracted polysaccharides by DEAE-cellulose DE-52.They were named HALWP-1,HALWP-2,HALWP-3 and HALWP-4.The yields of polysaccharides were 3.03%,4.19%,1.82%and 0.96%.HALWP-1,HALWP-2and HALWP-3 were shown to contain no protein and nucleic acid by UV-seanner.HALWP-4might be had protein structure.Infra-red spectrometry showed that HALWP-1,HALWP-2,HALWP-3 and HALWP-4 had typical absorption peaks of polysaccharides,speculated the functional groups were O-H,C-H,C=O,C-O and uronic acid,HALWP-1,HALWP-2,HALWP-3 and HALWP-4 might be?-pyranose.Their relative molecular mass were 3.82×10~5,2.98×10~5,3.36×10~5 and 4.72×10~5 Dal by using HPLC.3.This study chosed SGC-7901,HCT-8 and KB to identify the antitumor activity in vitro of extracted polysaccharide.The MTT method was used to determine the effect of inhibitory rate to these tumor cells.The results showed that the inhibitive rates of gastric carcinoma cell line SGC-7901,HCT-8 and KB were 50%with the water soluble polysaccharides from the wick of sunflower stem which were 4.201±0.083 mg/m L,5.843±0.069 mg/mL and 3.016±0.078 mg/mL respectively.These data showed that the antitumor activity in vitro of extracted polysaccharide were weak.These results were insufficient to explain the clinical antitumor activity.4.In order to study the antitumor effect in vivo of polysaccharide isolated from the wick of sunflower stem,we made the tumor mouse model by S180 tumor to Kuming mice.The results showed that the average weight of negative control groups(Stroke-physiological saline solution),CTX-treated groups(25 mg/kg)and polysaccharide-treated groups(250 mg/kg)were36.17±1.26 g,33.42±1.33 g and 35.06±1.19 g respectively.The inhibitory rates in CTX-treated groups and polysaccharide-treated groups were 63.73%and 51.16%respectively.The mean survival time of each groups were 49.58 d,54.08 d and 57.83 d respectively.The results showed that the side effect of the polysaccharide was small,it could prolong the survival time of KM mice significantly.5.In order to study the inhibiting tumor metastasis effect in vivo of four kinds of polysaccharide fractions,we made the tumor mouse model by transplanted lewis lung carcinoma to C57BL/6 mice.The results showed that the average weight of negative control groups,CTX-treated groups and four kinds of polysaccharide fractions treated groups were22.57±1.08 g,20.94±1.22 g,21.32±1.36 g,21.78±2.02 g,21.12±1.85 g,21.67±0.98 g and 22.23±1.34 g respectively.The inhibitory rates in CTX-treated groups and four kinds of polysaccharide fractions treated groups were 61.32%,52.17%,48.45%,45.12%,56.86%and58.61%respectively.The mean survival time of each groups were 38.7 d,40.8 d,47.3 d,43.1 d,46.2 d,47.7 d,50.5 d respectively.The results showed that the anti-tumor effect of four kinds of polysaccharide fractions were close to the CTX-treated groups,the side effect of four kinds of polysaccharide fractions were small,they could prolong the survival time of C57BL/6 mice significantly.6.In order to study the immunostimulatory of four kinds of polysaccharide fractions,we made the tumor mouse model by transplanted lewis lung carcinoma to C57BL/6 mice.The results showed that the spleen index of negative control groups,CTX-treated groups and four kinds of polysaccharide fractions treated groups were 6.53±0.52 mg/g,6.07±0.49 mg/g,7.08±0.38 mg/g,6.91±0.56 mg/g,7.04±0.67 mg/g,7.13±0.52 mg/g and 7.33±0.43mg/g respectively.The thymus index of each groups were 1.28±0.33 mg/g,1.19±0.29 mg/g,1.42±0.42 mg/g,1.39±0.37 mg/g,1.35±0.42 mg/g,1.46±0.38 mg/g and 1.52±0.42 mg/g respectively.Polysaccharide and its four fractions exerts their anti-tumor effect possibly through promoting the proliferation and differentiation of immune cells and as a result inereasing the immune effect or cells in the tumor-bearing mice.7.This study used enzyme-linked immunoadsordent assay(ELISA)to develop the effect of sunflower and its four kinds of polysaccharide fractions on the production of TNF-?.The results showed that the production of TNF-?in RAW264.7 cells of negative control groups,four kinds of polysaccharide fractions groups were 179.26±10.52 pg/m L,166.70±9.78pg/m L,170.79±10.36 pg/m L,175.06±10.56 pg/m L,173.51±9.33 pg/mL and 168.24±9.82pg/m L respectively.The production of TNF-?in serum of each groups were 163.23±8.26pg/m L,149.53±9.12 pg/m L,143.18±8.52 pg/mL,148.08±9.23 pg/m L,155.94±8.97pg/m L and 148.25±7.98 pg/mL respectively.The results showed that polysaccharide and its four fractions could reduce the TNF-?which was produced by macrophages.Polysaccharide and its four fractions which perhaps possess similar spatial structure to versican can competitively antagonized activation of TLR2 of macrophage.The amount of TNF-?would be reduced and the transfer of tumor cells would be postponed.All of these conditions set the foundation for development of the wick of sunflower to be the low toxicity of antitumor drug. |
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