| Helianthus annuus.L,the stem cores' the aqueous extractis used in the treatment of advanced malignant tumors in China,indicating that the aqueous extract of the stem core of the sunflower contains an antitumor active substance.In the process of searching for anti-tumor active substances in Helianthus annusus.L,stem cores,the stem core polysaccharides were screened out.Firstly,the structure of the stem core polysaccharide and the anti-lung cancer metastasis activity and mechanism of action were studied.The main findings are as follows:1.Preparation of Helianthus annuus.L,stem core polysaccharide:Firstly,the stem core polysaccharide was extracted by water extraction and alcohol precipitation method.The obtained crude polysaccharide was removed by the Sevag method,and the pigment was removed by activated carbon method.Then,the DE-52 ion exchange chromatography column was used for separation.Four elution peaks(HALWP1,HALWP2,HALWP3,HALWP4)were obtained.The anti-tumor activity of HALWP-4 on the Sephadex G-200 column was separated again to obtain four elution peaks(HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4),their The yields accounted for 2.09%,2.06%,3.05%and 0.66%of HALWP4,respectively.The average molecular masses of HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4 were determined by high performance gel exclusion chromatography to be 6.85 x 105 Da,5.42 x 105 Da,1.66 x 105 Da,1.03 x 105 Da,respectively.2.Preliminary analysis of the structure of the stem core polysaccharide:complete hydrolysis of the stem core polysaccharide(HALWP),derivatization and mass spectrometry,combined with infrared spectroscopy to speculate that the monosaccharide composed of the stem core polysaccharide may be ?-D mannose,?-D monosaccharide of mannose,?-D glucose,?-D glucuronic acid,(3-D galactose or ?-D galacturonic acid or a monosaccharide of aldehyde An acid or several monosaccharides and monosuuroic acids,and a certain amount of monosaccharides are covalently bonded to the fatty acid by glycerol.HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4 were completely hydrolyzed,and after derivatization mass spectrometry,combined with infrared spectroscopy and source speculation,HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4 were synthesized.The monosaccharide is(3-D mannose,?-D mannuronic acid,?-D-glucose,?-D glucuronic acid,?-D galactose or ?-D-galacturonic acid.A monosaccharide or a monosaccharide or a monosuuroic acid of a monosaccharide or a aldehyde thereof,and a certain amount of a monosaccharide is covalently bonded to a fatty acid or an organic acid by glycerin.3.In vitro anti-tumor activity of HALWP4:? The in vitro antitumor activity of the stem core polysaccharide HAWLP4 was studied by colony formation experiment:mouse lung cancer LLC cells were treated with 4.0 mg/mL different polysaccharides,and the number of colony formation in the drug-administered group was significantly reduced compared with the blank group.The number of cells in the blank group was 2×106,the inhibition colony formation rate in the HAWLP4-1 administration group was 52.17 ± 0.75%,and the colony formation inhibition rate in the HAWLP4-2 administration group was 56.52 ± 1.23%.HAWLP4-3 administration The colony formation inhibition rate of the group was 65.21 ± 0.98%,and the colony formation inhibition rate of the HAWLP4-4 administration group was 69.56±1.34%,n=3,P<0.05.?The ability of HALWP4-4,which inhibits tumor colony formation in 4.0 mg/mL HAWLP4,to inhibit tumor cell proliferation in vitro was evaluated by MTT assay:HALWP4-4 on human gastric cancer cell SGC-7901,human liver cancer cell L-02 and human oral epithelial carcinoma The IC50 values of cell KB were 3.978 ± 0.079 mg/mL,4.871 ±10.044 mg/mL,6.871±0.079 mg/mL,n=3,P<0.05.The results showed that the stem polysaccharide HALWP4-4 inhibited the proliferation of tumor cells in vitro,which was not enough to explain the strong anticancer activity of the aqueous extract of the stem.4.Anti-tumor metastasis activity of the stem core polysaccharide in vitro:? Transwell chemotaxis chamber was used to determine the inhibition of tumor cell migration by sunflower stem core polysaccharides:320 cells in the blank group,4 mg/mL sunflower stem core polysaccharides(HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4)has the effect of inhibiting LLC migration of mouse lung cancer cells.The cell migration rates of the four administration groups were 90.63 ± 0.49%,75.00 ± 1.32%,40.63 ± 1.07%,25.00 ±0.99%,n=3,P<0.05,n=3,respectively.P<0.05.Among them,HALWP4-4 has the most significant effect on inhibiting tumor cell migration.?Scratch test was used to determine the inhibition of tumor cell migration by sunflower stem core polysaccharide:the experimental results showed that the blank group,4 mg/mL sunflower stem core polysaccharide(HALWP4-1 group,HALWP4-2 group,HALWP4-3 group and HALWP4-The healing rates of the 4 groups were 23.05 ± 0.58%,17.25 ± 1.97%,13.86 ±0.98%,9.07 ± 2.03%,6.90±0.51%,n=3,P<0.05.The four components of sunflower stem core polysaccharide(HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4)have the ability to inhibit LLC metastasis of mouse lung cancer cells,and HALWP4-4 group inhibits LLC metastasis of mouse lung cancer cells.The most capable.5.Anti-tumor transfer mechanism of sunflower stem core polysaccharide:? The anti-inflammatory ability of sunflower stem core polysaccharide was studied by measuring the release of NO content in mouse macrophage Raw264.7.4.0 mg/mL HALWP4-1 group,4.0 mg/mL HALWP4-2 group,4.0 mg/mL HALWP4-3 group and 4.0 mg/mL HALWP4-4 group,respectively,the NO content was 11.35±0.21 ?mol/gprot,7.89±0.39?mol/gprot,7.69 ± 0.17 ?mol/gprot,7.57 ± 0.18 ?mol/gprot,n=3,P<0.05.The NO content decreased with the increase of drug concentration,so it has anti-inflammatory activity to four groups of Japanese sunflower stem core polysaccharides(HALWP4-1,HALWP4-2,HALWP4-3 and HALWP4-4),and HALWP4-4 The group has the strongest effect.? The effect of different components of sunflower stem core polysaccharide on the amount of TNF-? secreted by mouse macrophage Raw264.7 was detected by ELISA kit.The concentrations of TNF-? in the blank group,HALWP4-1 group,HALWP4-2 group,HALWP4-3 group and HALWP4-4 group were 179.26±10.52 pg/mL,166.70±9.78 pg/mL,and 175.06± 10.65 pg/mL,respectively.,173.51 ± 9.33 pg/mL and 168.24 ± 9.82 pg/mL.In summary,the sunflower stem core polysaccharide has antitumor activity and inhibits tumor metastasis activity.In addition,the sunflower stem core polysaccharide has an anti-inflammatory effect and inhibits the release of mouse macrophage TNF-?.Therefore,combined with the structural characteristics of sunflower stem core polysaccharides,it is speculated that the mechanism of sunflower stem core polysaccharides against lung cancer metastasis is:water-soluble sunflower stem core polysaccharide through its long-chain fatty acid and lung cancer tumor microenvironment macrophage Toll 2 receptor(TLR2)The binding of nearby cell membranes interferes with the binding of the multifunctional glycoprotein released by lung cancer tumor cells to the Toll 2 receptor(TLR2)of macrophages,thereby reducing the release of TNF-? by macrophages and ultimately inhibiting the metastasis of lung cancer.This study lays a solid theoretical and practical foundation for the development of sunflower stem core polysaccharides as a highly effective and low-toxic anti-tumor drug. |
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