Structural Characterization Of Polysaccharide DOPA-1 Isolated From Dendrobium Officinale Kimura Et Migo In Huoshan And Its Apoptosis Effect On HepG2 Cell

Dendrobium officinale,the dry stem of Orchidaceae plant Dendrobium officinale Kimura et Migo,which is one of the rare medicinal herbs in China,with the reputation of ‘the first of the nine Chinese immortals grass',has the effect of tonifying stomach and promoting fluid and nourishing Yin and clearing heat.Dendrobium officinale are mainly distributed in Anhui,Zhejiang,Hunan and Yunnan in China.Among them,Dendrobium Officinale Kimura et Migo in Huoshan is also called ‘wanzhangxu',which has been paid much attention by community as the authentic medicinal materials of Anhui Province,it is also regarded as the top grade of Dendrobium by ancient researchers.Dendrobium officinale contains polysaccharides,alkaloids,benzyl,amino acids and other active ingredients.polysaccharide as one of the main active ingredients of Dendrobium officinale,has the effects of anti-tumor,anti-aging and anti-oxidation.Therefore,in our study,the deproteinization method of the polysaccharide from Dendrobium officinale Kimura et Migo in Huoshan were evaluated,and the DEAE-52 cellulose column and Sephadex G-100 column were used to purify polysaccharides.Moreover,the structural characteristics and inhibition of the proliferation of HepG2 cells were further explored.1.Study on extraction process of polysaccharide from Dendrobium officinale in Huoshan.The crude polysaccharides were extracted by water extraction and alcohol precipitation,and the yield was 4.2% after freeze-drying.The optimal methods were evaluated in terms of the removing ratios of proteins and polysaccharide,the method of Enzyme-Sevag was found to be most effective for deproteinization,with optimum conditions: trypsin addition 0.5 mL,enzymolysis temperature 35 oC,enzymolysis time 40 min,with Sevag method extracted three times.In summary,the protein removal rates and polysaccharide loss rates were 81.58%,15.63%,respectively.2.Study on the purification and composition analysis of polysaccharide from Dendrobium officinale in Huoshan.The active polysaccharide DOPA-1 was isolated and purified by activated carbon,DEAE-52 cellulose column and Sephadex G-100 gel column;The UV and HPLC profiles demonstrated that DOPA-1 reached a certain uniform purity;The monosaccharide composition of the component DOPA-1 was analyzed by GC,the results indicated that DOPA-1 was mainly composed of mannose,glucose and galactose,the molar ratio of DOPA-1 was 1: 0.42: 0.27;The sugar content was 83.1%,uronic acid content was 2.31% of DOPA-1 by phenol-sulfuric acid method and carbazole sulfate method,respectively.3.Study on the physic-chemical characters and structural analysis of polysaccharide DOPA-1 from Dendrobium officinale in Huoshan.The physiochemical properties and structure of DOPA-1 were studied by HPLC,IR,NMR,periodate oxidation-smith analysis and other analytical methods.The results showed that DOPA-1 was white flocculus,tasteless,water-soluble and undissolved in organic solvents such as ethanol and acetone and there was no starch,amino acid or protein in DOPA-1.The specific rotation of DOPA-1 was + 128.19o,and the molecular weight was 2.29 x 105 Da.It was initially speculated that the main chain of DOPA-1 was a 1?3 bonded sugar group,branched to 1?2 position bonded glycosyl,1?6 position bonded sugar moiety was located at the end of the molecule.The analysis of IR spectrum and Cango-red indicated that DOPA-1 contained ?-D-mannopyranose and possessed a triple helix conformation.4.Study on the apoptosis effect on HepG2 cell of polysaccharide DOPA-1 from Dendrobium officinale in Huoshan.To study the inhibitory effect of DOPA-1 on the proliferation of HepG2 cells in vitro and to explore the possible mechanism.Our study revealed that DOPA-1 could inhibit the growth of HepG2 cells in a dose-dependent manner.Cell scratch test showed that DOPA-1 had a certain inhibitory effect on the migration ability of HepG2 cells.On the basis of the detection of mitochondrial function,it was found that DOPA-1 could injure the mitochondria of tumor cells by increasing ROS level and decreasing the mitochondrial membrane potential in HepG2 cells.The results of mitochondrial membrane potential showed that the permeability of mitochondrial membrane increased and the membrane potential decreased with the increase of DOPA-1 concentration,leading to apoptosis of HepG2 cells.The results flow cytometry showed that the apoptosis rate of HepG2 cells increased with the increase of DOPA-1 concentration,indicating that DOPA-1 could induce HepG2 cell apoptosis.Furthermore,the molecular mechanism of apoptosis induced by DOPA-1 may be related to down-regulation of Bcl-2 expression and up-regulation of Bax expression.