| Inonotus obliquus(I.obliquus)is a kind of rare medicinal fungi,with important research value.Many researches in medicine and pharmacology have proved that I.obliquus has certain therapeutical effects on anti-tumor,antioxidation,hypoglycemic effect and so on,and the main efficient components are polysaccharides.However,up to now,the studies on the polysaccharides from I.obliquus are still in the initial stage both at home and abroad.The anti-proliferative effects of polysaccharides from I.obliquus on colon cancer cells are also rarely reported.Therefore,we investigated the anticancer effects and its possible mechanism of polysaccharides from I.obliquus by ethanol precipitation and fractionated by ultrafiltration on HT-29 colon cancer cells,and this study to provide theoretical basis and guidance on the exploitation and utilization of polysaccharides.Six polysaccharide fractions(IOP40,IOP50,IOP60,IOP70,IOP80 and IOP90)were successfully isolated from I.obliquus by using different concentrations of alcohol precipitation.Then their physicochemical propertiess and anticancer effect were analyzed.Results suggested that IOP60 showed the highest yields,protein content and uronic acid content,while IOP40 exhibited the highest sugar content;six polysaccharides significantly inhibited the proliferation of HT-29 colon cancer cells in a time-and dosedependent manner(P<0.05).The order of anticancer activity were IOP60>IOP80>IOP90>IOP70>IOP50>IOP40,and IOP60 showed the strongest biological activity.The results of decolorization and deproteinization for IOP60 indicated that the activated carbon was the ideal decolorant for IOP60.IOP60 was identified as a glycoprotein by periodate-schiff staining.Five different molecular weight polysaccharides(IOP60a,IOP60 b,IOP60c,IOP60 d and IOP60e)were fractionated from IOP60 by ultrafiltration.Among the five polysaccharides,IOP60 b with the molecular weight range of 10 k Da-30 k Da possessed the most proportion and showed the strongest anticancer effect on HT-29 cells,and the sugar content of IOP60 b was 75.69%.As determined by HPLC,IOP60 b was composed of monosaccharides such as L-arabinose,D-ribose,D-xylose,D-galactose,D-glucose and D-mannose,and the molar ratio thereof was 12.33: 14.05: 1.29: 21.49: 26.06: 36.05,respectively.Then the anticancer effect and its possible mechanism of IOP60 b on HT-29 colon cancer cells were studied systematically.The cell morphology of HT-29 cells was significantly changed after IOP60 treatment by an optical microscopy,Hoechst 33258 fluorescence staining and hematoxylin-eosin staining.The results of DNA ladder experiments,PI-Annexin V staining experiments and cell cycle analysis showed that IOP60 b could significantly induce apoptosis of HT-29 cells and cell cycle was arrested at G0/G1 phase.The results of flow cytometry showed that the mitochondrial transmembrane potential of HT-29 cells was decreased after IOP60 b treatment.RT-PCR and Western blot were adopted to detect the m RNA and protein levels of apoptosis genes(Bcl-2,Bax and Caspase-3)after IOP60 b treatment.The results suggested that IOP60 significantly induced cellular apoptosis of HT-29 cells by upregulation the expression of Bax,Caspase-3 and downregulation the expression of Bcl-2,indicating that IOP60 might induce cellular apoptosis of HT-29 cells by mitochondrial pathway. |
