Study On The Extraction And Interaction With DNA Of Polysaccharides From Camellia Oleifera Abel Seed

In this work, the extraction, purification and structure characterization of polysaccharides from Camellia oleifera Abel seed were studied. The antioxidant ability in vitro, the protective effect against oxidative DNA damage and the interaction with DNA were also investigated. This study will lay the foundation in development and application research of polysaccharides from Camellia oleifera Abel seed in the cosmetics, pharmaceutical and health food industry. The main contents and results are as follows:The saponin from Camellia oleifera Abel seed was extracted with ethanol process as the pretreatment and the hot water extraction method was used to extract the polysaccharides from Camellia oleifera Abel seed. The optimum removal process of saponin was obtained as follows: ethanol concentration 95%, solid-liquid ratio 1:8 g/mL, extraction time 120 min, extraction temperature 55℃. Under the optimal conditions, the extraction yield achieved 10.97%. According to the results of single factor and orthogonal experiment, the optimum extraction conditions of polysaccharides were as follows: extraction time 3 h, extraction temperature 70℃, solid-liquid ratio 1:10 g/m L and the extraction yield was up to 30.31%.With removal of protein, chromatography of DEAE-cellulose to purify the polysaccharides from Camellia oleifera, three fractions were obtained and their identification and structural analysis were also investigated. In the protein removing process, the effect of deproteinization was compared among four methods including Sevage method, polyamide method, trichloroacetic acid method and enzyme method. The alkaline protease was used to deproteinize with the protein removal rate 74.38% and the retention rate of polysaccharides 75.40% respectively. After deproteinization, the yield of Camellia oleifera polysaccharides(TSP) was up to 5.62%. By DEAE-cellulose column, the crude polysaccharedes were further purified and three fined polysaccharides named TSP-1, TSP-2, TSP-3 were obtained with the yield 1.19%, 1.01% and 0.457 %, respectively. With Sephadex G-100 gel chromatography and HPGFC for further identification, it was found that TSP-1, TSP-2 had relatively homogeneous molecular weight distribution of 6760 Da and 6488 Da respectively while TSP-3 had a broad molecular weight distribution mainly of 7999 Da and 91862 Da. The glycopeptide linkage tpye of three polysaccharides was O-linked and both were pyranoside compounds. Besides, TSP-1 was mainly composed of glucose, TSP-2 and TSP-3 were both composed of arabinose, galactose and glucose.The antioxidant activity of polysaccharides from Camellia oleifera Abel seed was investigated by the methods of ·OH scavenging, H2O2 scavenging, DPPH·scavenging and the reducing power activity. Meanwhile, the protection capacity against DNA oxidative damage of Camellia oleifera polysaccharides was also studied. The results indicated that the IC50 values of TSP against ·OH, H2O2 and DPPH· were 5.47 mg/m L, 4.03 mg/m L and 0.54 mg/m L, respectively while the reducing power activity was quite low. In case of protective effect on oxidative DNA damage, the results showed that TSP had an effect with various levels on oxidative DNA damage induced by ·OH and H2O2. According to the fluorescence results, it was found that with the concentration of 0.20 mg/m L, TSP had the strongest protective effect against ·OH-induced oxidative DNA damage while with the concentration of 0.03 mg/m L, TSP could protect the H2O2-induced oxidatived DNA best. UV spectrophotometry result showed that with 1.5 mg/mL TSP, the protective effect of oxidatived DNA was up to 56.7%. The results of agarose gel electrophoresis indicated that with 0.04 mg/m L TSP, the protection was strongest with 66.9% of remaining supercoiling of DNA.Fluorescence spectroscopy and UV spectroscopy were employed to study the interaction between TSP and DNA. According to the fluorescence spectroscopy results, the fluorescence intensity of protein-combined TSP was quenched by DNA and the fluorescence quench process was the coeffect of dynamic quenching and static quenching. The binding constants Ka were measured and calculated at different temperatures with the values between 2.717×103 to 3.400×103 L·mol-1 and the binding sites were all about 1. By UV spectroscopy, ionic effect and KI effect, it was conjectured that the interaction between TSP and DNA was mainly through groove binding mode.