| Agaricus blazei Murill (ABM) has been consumed by humans not only as a part of food but also as a kind of medicinal materials due to its possible medicinal properties. At present, China is the largest exporter of ABM. And the content of polysaccharides from ABM ranked first among the edible fungi, and the research on ABM was benefit for its integrated utilization and development. In this paper, we conduct Agaricus blazei Murill as raw materials, via ultrafiltration to obtain low molecular weight polysaccharide, and then to study its isolation and purification, structure and anti-tumor activity. The results were as follows:1、In this study, the water-soluble polysaccharides were extracted by hot water form the the Agaricus blazei handle powder, deproteinized with papain and Sevag method, and ultrafiltrated by the hollow cellulose membrane module, in order to gather LAP which was short for the low molecular weigh ABM polysaccharide (10-100kDa) with a 21.18% recovery rate. And it was found the LAP contained 87.33% polysaccharide and 4.46% protein.2、The LAP was isolated by DEAE-52 ion-exchange chromatography, and its optimum elution conditions was established:NaCl gradient elution concentration was Omol/mL,0.05moL/mL, 0.1mol/mL, and 0.2moL/mL; the flow rate was 1.6mL/min; and the sample concentration was 20mg/mL while its volume was 5mL, equivalently to 5% of column volume. And under this condition, four polysaccharide fractions named LAP-1, LAP-2, LAP-3 and LAP-4 were obtained, and with a 8.09%,77.76%,9.62% and 4.54% distribution, respectively.3、The physics and chemistry character showed that the polysaccharides content of LAP-1-4 was 9.27%,90.92%,86.45%,67.63%, respectively, and LAP-3 and LAP-4 contained a small amount of protein, while LAP-1 and LAP-2 didn’t. It also suggested that the four fractions were kinds of non-starch polysaccharides without reducing sugar and phenols. The results of GC showed that LAP-1 was composed of arabinose, mannose, glucose and galactose; LAP-2 was mainly composed of glucose and galactose; LAP-3 and LAP-4 consist of glucose, as well as a small quantity of arabinose, mannose and galactose.4、The purity of the four fractions was identified by the method of Gel filtration chromatography. And the results showed that the four fractions are non-uniformitarian polysaccharides, and need to be further purified. Therefore, LAP-2 which was the main component of LAP was selected for further purification using Sephadex G100. And its optimum condition was followed:the concentration and volume of LAP-2 was 4mg/mL and 5ml, and the eluentic solvent was 0.15mol/L NaCl with 1.0mL/min flow rate. Under this condition, the sub-fraction polysaccharides named LAP-2-I, with a single component, was collected.5、LAP-2-I, a white flocculent powder, was found that it was soluble in water, and insoluble in ethanol, n-butanol, chloroform, acetone, and ethyl acetate. The 92.5% of LAP-2-I was polysaccharide, and it’s free form proteins and nucleic acids. In addition, it was turned to be a dextran with+19.3° optical rotation. The structure of LAP-2-I was characterized by infrared (IR) spectrum, gas chromatography (GC), periodate oxidation, and Smith degradation. And the results showed that LAP-2-I was α-D-glucopyranose, and mianly consisted of -(1')-linked,-(1'6)-linked,-(1'4)-linked, and -(1'4,6)-linked glucose, as well as a certain -(1'3)-glycosidic linkage, and a small amount of-(1'2)-,-(1'2,4)-glycosidic linkage. Besides, X diffraction and Congo red reaction also showed that LAP-2-I in an amorphous state didn’t had the crystal, polysaccharide complexes, or triple helix.6、In vitro, it was found that LAP, LAP-2, and LAP-2-I can inhibit the proliferation of SGC-7901 cells and promote apoptosis, with a dose-dependent relationship. However after multi-stage purification, their activities on inhibitation of proliferation and induction of apoptosis on SGC-7901 cells were reduced, which may be related to the synergy between the different components of LAP. And the relationship between the structure and acticities need to be more reseached.7、The anti-tumor activity for DLD-1 was also studied, and the result showed that LAP, LAP-2 and LAP-2-I can inhibit the proliferation of DLD-1 in certain extent, but not obvious, and they all had no induction on DLD-1 cell apoptosis. |
PDF Full Text Request