Inhibition Of Polysaccharides From Ganoderma Lucidum On Hepatocarcinoma Cells And Molecular Docking Between Polysacchairdes And Human Serum Albumin

Ganoderma lucidum is a class of traditionally Chinese precious herb. It exerts variousbiological activities including anticancer activity and immune regulation. Nowadays, thesubmerged fermentation of Ganoderma lucidum is a promising technology because it reducesproduction times, improves desired components and contents, solves the problem ofGanoderma lucidum scarce. In a word, this technology is benefit to deeply exploit and utilizeGanoderma lucidum. The polysaccharide from Ganoderma lucidum is viewed as one of themain pharmacological components and it can efficiently suppress tumorigenesis or inhibittumor growth. In addition, the anticancer activity of polysaccharide is related to its binding tohuman serum albumin (HSA), for HSA plays an important role in transport and deposition ofmedicine. Thus, the investigation of interaction between HSA and polysaccharide makescontribution to the exploitation, adjustment to dosage and pharmacology of polysaccharide.However, the interaction between HSA and polysaccharide has not been in literature and themechanism of anticancer activity of polysaccharide from fermentation of Ganoderma lucidumis seldom reported. Based on the facts above, the inhibition of polysaccharides fromsubmerged fermentation of Ganoderma lucidum on hepatoma cell line HepG2was studied inboth directly anticancer activity and immune regulation aspects. Subsequently, a miRNAmicroarray assay was employed to verify the miRNA profile from HepG2cells treated withthe intracellular polysaccharide. In addition, it is known that oxidative DNA nucleotide is akind of typical oncogene that the oxidation occurs at5’ end of DNA. Meanwhile, thesequence of miRNA on the chip surface is3’endâ†'5’end. Therefore, the oxidation of DNAnucleotide affects the hybridization by influencing the base stacking and base paring. Tounderstand the effect of oxidative DNA nucleotide on pairing between DNA-RNA (miRNA)complex, the interaction between DNA nucleotideand RNA nucleotide was investigated byquantum chemistry using Guassian program. Furthermore, the molecular docking betweenpolysaccharide and HSA was also investigated using ZDOCK module in Discovery studio2.5.It can predict the anticancer activity of the polysaccharide in terms of its binding affinity toHSA.The extracellular polysaccharide (GLP-Ex) and intracellular polysaccharide (GLP-In)were respectly extracted from supernatant and mycelia of submerged fermentation ofGanoderma lucidum. According to weight-average molecular weight (Mw) andmonosaccharide component, the main part of GLP-Ex has the Mwof9074Da with the molarratio of the monosaccharides of Fuc: Glucosa: Ara: Galactosa: Gal: Glu: Xyl: Man=1.00:0.19:6.16:0.11:12.03:83.08:10.05:3.42; GLP-In contains Ara, Gal, Glu, Xyl and Man in amolar ratio of1.00:2.33:40.96:2.59:2.03. In addition, the effect of the two polysaccharideson HepG2cell line was studied by MTT method. The result shows that the inhibition rates ofGLP-Ex and GLP-In on HepG2cell line were7.55%and29.52%at the concentration of500μg/mL for48h. Meanwhile, the effect of GLP-Ex and GLP-In on nitrite production byRaw264.7macrophages was also investigated. It is indicated that GLP-In was more preferableto stimulate Raw264.7cell to produce NO than GLP-Ex and the two polysaccharides had nodirect cytotoxic effect on Raw264.7cell. To understand the anticancer mechanism of the polysaccharide, miRNA profile from HepG2cells treated with the GLP-In was verified usinga miRNA microarray assay. It is shown that61miRNAs had differential expression,17ofthem were regulated significantly. Moreover, miRNA-3131represented the strongestupregulation with92fold. The miRNAs responded synergistically in both hepatocarcinomaand immune-related aspects.Oxidation of DNA is one of the main factors to induce cancer. Oxidative DNAnucleotide affects the pairing between DNA and RNA (miRNA) during hybridization on chip.Based on the fact above, the interactions between oxidative2’-deoxyadenosine nucleotides(2OHA,8OHA,8OXOA, fapyA) and canonical ribonucleotides (A, C, G, U) wereinvestigated. The result indicates that syn8OXOA: G complex is the most stable among allthe complexes discussed in this thesis. Furthermore, the hydrogen migration occurs in s8OHA:C complex to form more stable one.GLP-Ex presents weak anticancer activity. Our previous study shows that the sulfation ofGLP-Ex can improve its anticancer activity due to the change of conformation ofpolysaccharide. Thus, the biological activity of polysaccharide is closely related to itsconformation. In this thesis, the model polysaccharides were constructed according to themonosaccharide of GLP-Ex employing Discovery studio2.5(DS2.5) in Builder program.The main chain of the polysaccharides were composed of α-D-Glucan (α-D-Glc),α-D-Galactose (α-D-Galc), α-D-Mannose (α-D-Man) and the (1â†'6) and (1â†'3) linkedresidues were also taken into account. The sulfate groups were introduced into the modelpolysaccharides. It is shown that α-(1â†'3)-D-Glc trends to exist as linear conformation. Assulfate substitution happens at C4or C6position, this polysaccharide also takes a linearconformation. Other sulfated (1â†'3)-α-D-Glcs are in the forms of random coil conformations.(1â†'6)-α-D-Glc,(1â†'3)/(1â†'6)-α-D-Galc and (1â†'3)-α-D-Man take random coilconformations while most of their sulfated derivatives exist as linear conformations.(1â†'6)-α-D-Man possess a linear conformation of which chain is flexible. Except the sulfate groupat C3position that makes the chain in the form of random coil conformation, all the othersulfated derivatives take linear conformations. It is indicated that intramolecular electrostaticrepulsion makes the sulfated polysaccharides more extended and stiffer.The interaction between polysaccharide and HSA affects the anticancer activity ofpolysaccharide. The molecular docking between polysaccharide and HSA was investigatedusing ZDOCK module in DS2.5. It is shown that the unmodified polysaccharide has higheraffinity of binding to HSA than its sulfated derivatives. Thus, it can be predicted that thesulfated polysaccharide is easier to be released from HSA, enhancing its free concentration.As a result, it is benefit to exert the activity of this polysaccharide. Furthermore, based on themonosaccharide of GLP-Ex, the model disaccharides were constructed and their interactionswith HSA were also studied by CDOCKER module in DS2.5. These disaccharides exhibitdifferent binding affinity to HSA at different binding sites. In most cases, the complexes ofdisaccharides and HSA are more stable at the binding sitesof3and5position.