Study Of Structure And The Mechanism Of Antitumor Activity Of Morchella Esculent Polysaccharide

The current study is focused on chemical components and nutritional value of polysaccharide. With the development of separation and purification technology in recent years, the study on structural analysis is getting much concern. Research finds the polysaccharide extracted from Morchella esculenta were rich in bioactivities like anti-bacteria, anticancer, immuno-stimulant ect. However, the detailed mechanism is still unrevealed. This experimental studied the isolation, purification, property identification and antitumor mechanism of polysaccharide from Morchella esculenta. Base on researching the dependence of bioactivities on the structure of Morchella esculenta polysaccharides was understood. This will contribute to the development and application of polysaccharide from Morchella esculenta.In experiment, using response surface method optimization of cultivation conditions for fermentation of extracellular polysaccharide from Morchella esculenta. The low production of polysaccharides and waste of resources were been overcome. Then the structural characteristics of the purified polysaccharide from Morchella esculenta, and the polysaccharide induced cells apoptosis mechanism were investigated.(1) In the first step, a Plackett-Burman design was used to evaluate the effects of variables factors and statistically significant factors were screened out. Then, the optimal combined conditions for maximum polysaccharide production were further optimized by response surface methodology. Results:The optimal fermentation conditions were determined as follows, glucose 30%, cultivation time 5.98d, rotary speed 217.44rpm. The average yield of polysaccharide validation experiments was 53.04%, the relative error was 1.64%.(2) Crude water-soluble polysaccharide (MEP) was extracted from Morchella esculenta using hot water and ethanol precipitation. Then it was purified by DEAE-Sepharose F.F column chromatography. Total sugar content of the elution was determined by the phenol-sulfuric acid method. After that, the fraction was subjected to Superdex-200 column with AKTATM Purifier 10 system. Finally two polysaccharide fractions MEP-Ⅰand MEP-Ⅱwere obtained. High performance gel permeation chromatography (HPLC/ELSD) analysis showed the average molecular weight of two polysaccharides. Structural properties and compositions of these two fractions were examined by Fourier transform infrared (FT-IR) and a high-performance liquid chromatography evaporative light scattering detection (HPLC/ELSD). Results demonstrated that the average molecular weight of MEP-Ⅰand MEP-Ⅱwere approximately 3.91×103 and 1.58×105 Da. The two polysaccharides both contained mannose, galactose and glucose. But the molar ratio was different from MEP-Ⅰand MEP-Ⅱ. Glucose was the main sugar unit, accounting for 84.64% and 78.39%, additional to rhamnose and arabinose were also seen in the samples. IR spectrum demonstrated several bands corresponding to carboxyl group, which was consistent with the results of chemical methods. According to the analysis of wearing curve by tests and wear topography by SEM, polysaccharide existed in either a globular or a circle form. The ball and ring-like structure can be further connected together in a chain or side network structure.(3) MTT method was used to study the inhibition effect of polysaccharide from Morchella esculenta on HeLa, HepG2 and AS49 cells. HepG2 cells were selected effect most apparent in the tumor cells. First, in experiments apoptosis was proved by electron microscopy. Apoptotic morphology and apoptotic body of HepG2 cells were observed under fluorescent microscope and transmission electron microscope (TEM). Results suggested MEP could suppress proliferation and induce apoptosis in HepG2 cells. To study the variation of some apoptosis inducing factor expression in HepG2 cells the process of apoptosis induced by MEP. Apoptosis of the cells was detected by flow cytometry with AnnexinV-FITC/PI double staining. ROS was monitored by the oxidation-sensitive fluorescent dye DCFH-DA. The changes of mitochondrial membrane potential by examining the retention of Dioc6(3) were examined. There was a time-dependent decrease of Dioc6(3) fluorescence after treatment with MEP compared with the untreated control. MEP may induce apoptosis of HepG2 cells through mitochondrial permeability transition which decreased the△Ψm and released AIF from mitochondria.