| Inonotus obliquus belongs to the family of polyporaceae of basidiomycetes. It is mainly found at latitudes of 45°N-50°N such as North America, Finland, Poland,Russia, Heilongjiang and Jilin province in China(Chang Bai Shan mountain) and Japan et al. It has no toxic effect with anti-tumor, curing diabetes,preventing AIDS,risisting caducity and immunostimulation. Polysaccharides from Inonotus obliquus have many biological activities of anti-tumor, cutting down blood sugar and blood fat, immunostimulation and anti-oxidation..At present,study on Inonotus obliquus and its polysaccharides in china are unsubstantial.There is little report on isolation,purification,structure and mechanism of antitumor activity of polysaccharides from Inonotus obliquus. In this paper, based on extraction and preliminary purification of polysaccharides from Inonotus obliquus, physical and chemical character and antitumor activity of IOP were investigated. IOP was further isolated and purified. Single component named IOP3a which has strong antitumor activity was selected. Structural feature of IOP3a, the mechanism of antitumor activity both in vitro and in vivo and the molecular mechanism on apoptosis of tumor cells were also investigated. The main results of result are as follows:The chemical composion of Inonotus obliquus were analyzed:3.5±0.36% of moisture, 2.4±0.44% of protein,1.7±0.25% of crude fat,10.4±0.44% of ash,67.5±0.95% of crude fiber, 4.2±0.30% of reductive sugar,10.3±0.40% of polysaccharide.The optimal conditions for classical water extraction of IOP by orthogonal array were as follows:the extracting temperature was 90℃, the extracting time was 4h, solid/water ratio was 1:30 (W/V).Response surface methodology (RSM) was employed to optimize the ultrasonic/microwave assisted extraction (UMAE) conditions. The results indicated that the optimal conditions for UMAE were 90W microwave power, solid/water ratio was 1:20 (W/V) and the extracting time was 19min, respectively. Compared with traditional water extraction, the yield of polysaccharides increased from 2.12% to 3.25% at shorter extraction time (19min). The purity of polysaccharides increased from 64.03% to 73.16%. Structure of IOP extracted with two methods was the same basically by IR spectra analysis, which confirmed the ultrasonic/microwave assisted extraction of IOP was superior to classical water extraction. It was proved that polyamide was the best declor and deprotein methods on IOP.Physical,chemical character and antitumor activity of IOP were investigated.The results showed that dried IOP was light coffe color with no taste, easy to dissolve in water and could not dissolve in ethanol, methanol and acetone et al. The content of polysaccharides and uronic acid was higher with no protein. IOP consisted mainly of rhamnose,arabinose, xylose, manose, glucose and galactose, mol ratio of which was 10.25:9.38:1:12.45:9.9:11.55.Its molecular weight was 156611Da determined by HPLC. The viscosity of IOP increased with the increase of temperature step by step in a certain range of temperature. pH had no obvious effect on viscosity of IOP, metal ions had obvious effect on viscosity of IOP, Ca2+ and Mg2+ had great influence on viscosity, Na+ had the least influence on viscosity. Urea with low concentration could increase the viscosity,however, Urea with too high concentration could decrease viscosity. The result of antitumor activity both in vitro showed that IOP could kill jurkat and daudi tumor cell direcctly in vitro. The highest inhibition rate was 62.29% and 66.42%, repectively, which showed an excellent dose-response relation. At the same, IOP had obvious anti-tumor activity in vivo. The inhibition rate at low and high dose were 43.52% and 57.48%, repectively. IOP increased obviously the spleen index and proctected immune organ effectively. IOP improved macrophage phagocytosis in jurkat tumor-bearing nude mice.IOP was fractionated by anion-exchange chromatography (DEAE-Sepharose-CL-6B). Four components were gained. IOP1 was neutral acid polysaccharide. IOP2, IOP3 and IOP4 were acid polysaccharides.After fractionation of IOP, the recovery rate of polysaccharides was 85.6%. IOP1 and IOP3 were chosed because of their higher anti-tumor activity.IOP3 and IOP1 were eluted on sepharose CL-6B column. IOP3a and IOP3b as well as IOP1a and IOP1b with different molecular weight were gained. IOP3a and IOPlb were chosed because of their higher anti-tumor activity. Gel permeation chromatography, high-performance liquid chromatography (HPLC) and agarose gel electrophoresis indicated that IOP3a and IOP1b were homogeneous.The average molecular weight of IOP3a and IOP1b was 44265Da and 153172Da, respectively.Monosaccharide of IOP1b was glucose determined by gas chromatography, which was estimated to beβ-glucan.The antitumor activity and its mechanism of IOP3a were examined and evaluated with the nude mice transplanted jurkat tumor in vivo. At the high and middle dosage, IOP3a had stronger antitumor. activity with inhibition rate of 69.28% and 57.62% with an excellent dose-response relation compared with the control group (P<0.05).IOP3a could increase the spleen index,white blood cell and lymphocyte with the increase of dosage.The histopathology of tumors from the various groups indicated that the tumor cells from the different IOP3a treated groups had clear necrosis areas in tumor tissues.The expression of tumor gene Bax and Bcl-2 in tumor tissues between the experimental and control groups detected with immuno-histochemistry indicated that the antitumor activity of IOP3a in vivo was by the means of improving the expression of tumor gene Bax and inhibiting the the expression of tumor gene Bcl-2.Microscope, fluorescence microscope, scanning electron microscope, Flow cytometric and western blot were used to investigate apoptosis mechanism of jurkat and daudi induced by IOP3a in vitro. Results showed that the tumor cells treated with IOP3a for 48h showed significant morphological changes, such as cell shrinkage, distribution nonuniform and shape irregularity. Typical apoptotic morphological features (chromatin condensation with light blueness) were also observed by fluorescent microscopy.Scanning electron microscope (SEM) photos showed that the villus of the jurkat and daudi cells membrane was disappeared and broken off to form apoptotic bodies.FITC-Annexin V/PI staining with flow cytometric analysis showed that the apoptotic rate of Jurkat and Daudi cells reated with IOP3a for 48h was 16.8% and 23.1%,respectively, which was more than that of controll group(4.6% and 3.6%,respectively).The release of Cytc and expression of caspase-3 were demonstrated with western blot. At the same time, DNA ladders gel electrophoresis was carried out.The results showed that cell apoptosis mechanism induced by IOP3a was to induce cyt c release and caspase-3 activation via mitochondfia-controlled apoptotic pathway to activate endonuclease and cause DNA fragmentation.The structural characterizations of IOP3a were studied using methylation analysis methods combined with GC-MS,NMR and FT-IR testing instrument. The results indicated that IOP3a contained the main part of homogalacturonan fragments with→4)-α-GalpA (1→and rhamnogalacturonan segments with→4)-a-GalpA (1→2)-a-Rhap (1→backbone.→4)-β-Galp (1→and→6)-β-Glcp (→,→5)- -α- Araf(1→were linked as galactan,glucosan and arabinan, which were the mainly constitution of the branch, and linked to O-4 of 1,2-linked rhamnosyl residues.→6)-β-Galp (1→was linked as galactan, which were substituted at O-4 position and O-6 position.→5)-α- Araf(1→were linked as arabinan, which were substituted at O-3position.Conformation of IOP3a was determined tripe helical by congo red experiment.The result of ultra-violet spectrograph showed that temperature had no obvious influence on conformation of IOP3a. Acid, alkali and metal ions could change the tripe helical conformation in a degree. Thermogravimetic analysis showed that IOP3a decomposed and lost weight rapidly between 170℃to 500℃.
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