Extraction, Purification And Structure Elucidation Of LT1, A Polysaccharidesfrom Lentinus Edodes

Polysaccharides from Lentinus edodes is one of the most important biomacromolecules. It attracts much attention since 1960 for its immunomodulating effects. In recent years, many researches indicated that some polysaccharides isolated from L. edodes exhibited effects on anti-virus, anti-tumor, regulating the immune system, lowering blood sugar, antioxidant and other functions. LT1 was a novel polysaccharide isolated from L. edodes with molecular weight of 642 kDa and we found it reach the criteria of National Drug Standards WS1-320(X-263)-2004Z. This paper summarizes the results of chemical structure and chain conformation studies on LT1, mainly including the extraction and purification of LT1; the quality control of LT1; the chemical structure of LT1and the chain conformation of LT1.PART ONE THE EXTRACTION AND PURIFICATION OF LT1The methods for extraction and purification of polysaccharides from L. edodes were studied, so as to improve the conditions of isolating single molecular weight polysaccharides from L. edodes. Then a novel polysaccharide LT1(Mw=642 kDa) was obtained. The viscosity properties of LT1 were studied with capillary viscometric measurements. The viscometric measurements show moleculer association of LT1 in aqueous solutions. The results of molish test and KI-I2 test indicated LT1 is different from the structure of amylum.PART TWO STUDY ON QUALITY CONTROL OF LT1Quality control of LT1 was studied by national lentinan quality standard ( NO. WS1-320(X-263)-2004Z). FT-IR, UV-VIS and HPLC were performed to quantify its total carbohydrate content and characterized its chemical structure. FT-IR showed LT1 contains bothαandβtype residues. Total carbohydrate content quantified by the phenol–sulfuric acid method as 98.7%. The difference of absorption before and after smith-degradation was 0.19. All the results indicated LT1 confirming the national lentinan quality standard ( NO. WS1-320(X-263)-2004Z ). PART THREE THE CHEMICAL STRUCTURE OF LT1The chemical structure of LT1 was studied. Gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FT-IR), methylation analysis, HIO4 oxidation-Smith degradation, 1D and 2D NMR (DQF-COSY, HSQC, and HMBC) experiments were performed to characterize the chemical structure of LT1. The GC analysis result shows that LT1 only contained D-glucose. The FT-IR result shows that LT1 was consisted of bothαandβtype glucose. Based on the results of methylation analysis, HIO4 oxidation-Smith degradation and NMR spectra, a proposed chemical structure of LT1 was established:PART FOUR THE CHAIN CONFORMATION OF LT1The order–disorder transitions of a helix are often accompanied by some changes, which can be monitored by optical rotation. The NaOH weight fraction (WNaOH) dependence of [α]25D for LT1 in water–NaOH mixtures at 25℃were measured. Congo Red test was used to determine the chain conformation of LT1 in 0.1M NaOH solution. Atomic Force microscopy(AFM) was used to observe the LT1 molecule in aqueous solution and measure the thickness of it. [α]25D sharply decreases when WNaOH increases from 0.20M to 0.30M indicating that the helix-coil conformation transition of LT1 may carry out when NaOH concentration was higher than 0.20M. Compared with a pure Congo Red solution, Congo Red-glucan complex exhibiting a large red shift inλmax indicates that the existence of triple helix of LT1 dissolved in NaOH solution(0.1M).From the AFM image of the LT1 sample in distilled water, multiple molecules of thick rope-like structures can be observed. The thickness of the lentinan measured by AFM is about 2.20 nm averaged over hundreds of molecules which is approximately to the triple-strand thickness of a lentinan molecule with 1.73nm measured by X-ray diffraction . Some linear and several branched structures were also observed in image.Based on all the studies in this paper, we can conclude that LT1 is a novel polysaccharide isolated from L. edodes. It has a linear structure with a backbone composed ofα-1,4-linked,β-1,4-linked andβ-1,3-linked glucopyranosyl units and slightly branched at C-6 ofβ-1,4-linked glucopyranosyl by side chains, mainly as a single glucosyl stubs. Studies on chain conformation of LT1 show that LT1 exsits as triple-helix in distilled water and a helix-coil transition may occur when NaOH concentration is higher than 0.20M.