Isolation And Purification, Structural Investigation And Antitumor Activity Of Polysaccharide From Termitomyces Albuminosus Fruiting Bodies

Termitomyces albuminosus has very high value because of containing various nutrimentsand medical components. Now, the research hotspot of Termitomyces albuminosus focuses ondomestication and cultivation and mycelium cultivation. The research of Termitomycesalbuminosus polysaccharide lingers on extraction technology at present; there are few studieson structure analysis, structure-activity relationship and special function. Extraction, isolation,purification, primary structure and the antitumor activity of Termitomyces albuminosuspolysaccharide were systematically researched in the dissertation. The main contents andconclusions are listed as follows：The content of crude protein, total polysaccharide, crude fat, crude fiber, ash and water inthe dry sample were：32.82%,26.79%,4.06%,4.04%,6.38%,7.12%and13.08%.The extraction technology of TAP was investigated and extraction process was optimized.From single factor experiment, the best extraction temperature,liquid-solid ratio andextraction time were80℃or90℃,30:1,3h, respectively. In these conditions the second andthird yield were1.01%,0.39%, respectively.On the basis of single factor experiment, theextraction process was optimized by response surface methodology. The optimum conditionsmodified were as follows：extraction temperature87℃,extraction time2.9h,liquid-solid ratio31:1（mL/g）,the yield could come to16.88%in these conditions.TAP was made after polysaccharide extracted from Termitomyces albuminosus in theoptimum conditions was decolored,ethanol subsided and deproteinized.TAP was separated byDEAE-52ion-exchange column chromatography and centrifuged by100KD ultrafiltrationcentrifugal tube.Then two purified was made named TAPⅠ,TAPⅡ-2and one mixturecontaining TAPⅡ-1,TAPⅡ-2.TAPⅠ,TAPⅡ-2were identified as homogeneous componentby means of gel permeation chromatography and Sepharose CL-6B gel columnchromatography. The weight average relative molecular mass of TAPⅡ-1,TAPⅡ-1and TAPⅡ-2were14656,16544and1958484relatively.The structure of TAPⅠ was analysed. There was no protein and nuclear acid via UVlight scanning at260nm and280nm. The infrared spectra of TAPⅠ showed TAPⅠ waspolysaccharide,because TAPⅠcontained the stretching vibration of O-H at3412cm^-1, thestretching vibration of C-H at2930cm^-1, the antisymmetric vibration of C=O at1651-1，thestretching vibration of a group of carboxylic at1422cm^-1. TAPⅠ contained pyranoid ring and mannose. Monosaccharide composition analysis of TAPⅠshowed it was consisted offucose, mannose, glucose and galactoside, their mole ratios was1:1.38:2.47:3.08. Periodateoxidation and Smith degradation showed TAPⅠcontained1→6,1→,1→2,1→4and1→3typeglycosidic bond. All fucose, very large amounts of galactoside and part of glucose could beoxidized, the glycosidic bond was1→6or1→or1→2or1→4. Mannose and small amountsof glucose couldn’t be oxidized and should be1→3type glycosidic bond. Nuclear magneticresonance spectrum showed TAPⅠcontained α,β-configuration and was consisted of6kinds of sugar residues.Antitumor activity of polysaccharides from Termitomyces albuminosus fruit wasdetermined on two kinds of cancer cells，human hepatoma Hep G2cells and human bladdercancer T24cells by MTT assay. The results showed that TAP, TAPⅠ,TAPⅡ-2and mixtureof TAPⅡ-1and TAPⅡ-2had inhibitory effects against human hepatoma Hep G2cells in adose-dependent manner. The inhibitory effect of pure polysaccharide,TAPⅡ-1, was mostobvious and the inhibition rate could come to64.01%. All polysaccharides had low inhibitoryeffect on human bladder cancer T24cells. Only part of polysaccharides appeared inhibitioneffect in low concentration.