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Research of Correlation between Antitumor Activity and Structure of Lentinan

Author: WangJun
Tutor: WangKaiPing
School: Huazhong University of Science and Technology
Course: Pharmacy
Keywords: chemical structure NMR spectrum congo red atomicforce microscope (AFM) X-ray diffraction (XRD) antitumor activity
CLC: R284
Type: Master's thesis
Year: 2012
Downloads: 38
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Abstract


Lentinan has always been used for the adjuvant therapy of cancer as animmunomodulatory agent in the clinical. But recent studies have shown that ithas an effect of killing tumor cells directly as well. No matter in which ways, itindicated that its antitumor effect was closely related to the structure accordingto existing research results. Morever, the relationship between its structure andantitumor activity is still controversial at present. In this study, we extracted thecrude polysaccharide by water extraction-alcohol precipitation and alkalineextraction-alcohol precipitation from lentinus edodes. After decolorization,removing protein and ultrafiltration, five purified polysaccharide fractions areobtained, which named LST1、LST2、LJT1、LJT2and LJT3respectively. Weused a combination of chemical analysis, spectral analysis and moderninstrumental analysis techniques to identify the primary chemical structure andthe senior carbohydrate chain conformation, and also to discuss its antitumoractivity in vivo and vitro. Therefore, we studied the intrinsic link between itsstructure and antitumor activity to lay the foundation for further quantitativestructure-activity relationship studies and computer simulations of carbohydratechain conformation, and to provide theoretical and material basis forsubsequent molecular mechanisms of antitumor effect.Part I The extraction、separation and purification of LentinanFirstly, we extracted crude polysaccharide by water extraction-alcoholprecipitation and alkaline extraction-alcohol precipitation from lentinus edodeswhich was purchased from qingyuan, Zhejiang province. After decolorizationand ultrafiltration, five different and refined lentinan fractions with uniformmolecular weight that named LST1、LST2、LJT1、LJT2and LJT3were obtained.The sugar content and UV scanning were used to identify the samples’ purity,while by determination of molecular weight and specific rotation detection toanalysis their physical and chemical properties. The relative molecular weight of the five components were524kDa、65.5kDa、697kDa、200kDa and98.4kDa respectively by HPGPC. Besides, we measured the sugar content, which were97.94%、100.16%、101.37%、107.29%and103.99%by Phenol-sulfuric acid method. UV scanning showed that they didn’t contain nucleic acid and protein, and the results of the specific rotation detection were22.6、-10.4、10.6、3.4and-2.6. In short, the five refined components obtained not only have good traits and high purity, but also meet the requirements of the follow-up experiments.Part Ⅱ Study on the primary structure of LentinanTo determine the monosaccharide composition of different lentinan fractions by complete acid hydrolysis. Periodic acid oxidation and Smith degradation analyzed the type and proportion of their sugar residues. Methylation analysis further confirmed the sugar residues’ratio. Infrared spectroscopy scanning indicated the characteristic absorption peaks and the sugar residues’configuration of each purified polysaccharide component. NMR analyzed their sugar residue type, the connection sequence and substitution sites, which verified each other with the results of chemical analysis. The experimental results have showed that LST1、LST2、LJT1、LJT2and LJT3were composed of glucose, and the chemical analysis indicated that each of them contains four types of sugar residue, the proportion of them were (1-6and end:1-3and1-3-6) about1:1.6,1:1,1:3,1:2and1:2. IR scanning also showed that they had characteristic absorption peaks of polysaccharide, and the structure was β-D-pyranose. NMR detection further speculated that the main chain structure of each component was β-(1→3)-D-pyran-glucan while had branches at C6and the basic structural units were similar. Part Ⅲ Study on the sugar chain conformation of LentinanTaking LST1for example, the helical structure was disintegrated when treated with NaOH and would be refolded after dialyzed with distilled water. Then freeze-dried and renatured polysaccharide was obtained that named LSTls. Comparied the change of maximum absorption wavelength λmax that contributed by the complexes formed between the five fractions with congo red respectively under the condition of different concentrations of NaOH solution through congo red experiments. Therefore, inferred the helical conformation of the sugar chain of polysaccharide fractions and also investigated the space structure changes among LST1before degeneration and after and LST1s. Observed the surface morphology distribution of the five fractions、LST1s and denatured LST1by AFM and speculated their spatial conformation. XRD experiments detected the purified polysaccharide chain’s regular arrangement of the five fractions and also examined their crystallographic information. Experimental resultspreliminarily verified that the natural lentinan structure contains triple helicalconformations, whereas LST1s may be single-stranded helical structure.Furthermore, natural lentinan was in the form with linear branching in aqueoussolution. However, denatured LST1was spherical and LST1s was restored in alinear structure. The variations of the structure and the form suggested that thelentinan could still restore its helical conformation after denaturation with highconcentrations of NaOH. The sugar chain height of the natural lentinan matchedwith the crystal parameters (crystallite size) measured by XRD, which wereabout1.7nm. Therefore, all these could further confirm that each fraction hadtriple-helical spatial conformation. While the sugar chain height of LST1scombined the congo red experimental results, so it could be speculated thatLST1s was mainly in the form with single helical conformation.Part Ⅳ Research of antitumor activity of lentinan in vitro and in vivoInvestigating natural lentinan LST1、LST2、LJT1、LJT2and LJT3respectivelyinhibited proliferation of tumor cells in vitro by MTT assay, while LST1s inhibitedproliferation of H22cells in vitro. By selecting LST1for antitumor experiments invivo, further studies had been done to investigate the inhibition of lentinan againstproliferation of S180in vivo and its impact on the host’s immune system. Theexperimental results showed that all of lentinan fractions had inhibition against H22、S180and HepG2tumor cells, and showed good dose dependence, while the inhibitoryrate of high molecular weight polysaccharide was higher than that of low molecularweight polysaccharide; but all fractions were not sensitive to K562tumor cells,suggesting that the inhibitory effect of them had some selectivity for different tumorcells. The antitumor test results of LST1s in vitro preliminarily indicated that lentinanwith a single helical structure also had good antitumor effect. The antitumor testresults of LST1in vivo revealed that the low, middle and high dose groups hadinhibitory rate of54.03%,61.68%and53.52%, respectively. The increase in spleenindex and the number of immune cells indicated that LST1could stimulate the proliferation and differentiation of host’s immune tissue, and have a role in regulationof the immune. Combining with the results in vitro, it could be inferred that lentinanmight have dual antitumor effect, including directly kill tumor cells and immuneregulation, while it had fewer clinical side effects of chemotherapy drugs.Part Ⅴ The relationship between antitumor activity and structure ofLentinanCombining the these results and literature, this part would conduct in-depthstudy about the intrinsic link between antitumor activity and structure of lentinan fromfive aspects include main chain, molecular weight, degree of branching and branchlength, helical structure and water-solubility. The study concluded the vast majority ofnatural lentinan having the basic skeleton of β-(1→3)-D-glucopyranose, which is thepremise of its antitumor activity. The antitumor effect of high molecular weightpolysaccharides is stronger than that of low molecular weight polysaccharides whichare extracted by the same method. The branched side chain contributed to the stabilityof helical structure of sugar chains, on the other hand, the degree of branch couldexplain why high molecular weight lentinan had stronger antitumor effect, so that DBhad become an important factor to measure the antitumor effect. Single helicalstructure in the natural lentinan is the foundation of its biological activity, while triplehelical structure may be auxiliary role, so that lentinan could be recognised moreeasily by its receptor in vivo to activation of the immune system; combining with itsdirect inhibition of tumor cells, lentinan could play a dual antitumor effect. In addition,the solubility of lentinan in aqueous sulution is a factor to affect its antitumor effect.The exposition of the structure-activity relationship laid a theoretical foundation forthe next research on molecular mechanism.

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