Studies On Preparation, Components Analysis And Antitumor Activities Of Polysaccharides From The Fruit Shell Of Camellia Oleifera Abel

China has the largest camellia oil production in the world.Camellia oleifera distributes mainly in hilly regions of Hunan,Jiangxi and southwestern China and occasionally distributes in southeast Asia and Japan.Camellia oleifera is the special oil plant of China.The fruit of Camellia oleifera Abel is composed of fruit shell and seed,the contents of them are 63.64%and 36.36%, respectively.The camellia oil has high nutrition value on accounted of oleic acid and linoleic acid contents being more than 80%.The fruit shell of Camellia oleifera Abel can be used to extract tannin extract,furfural and xylitol.And it also can be turned into activated carbon and potassium carbonate.China is rich in Camellia oleifera Abel resources and the research about active materials from the fruit shell is rare.Recently,there are many researches about the bio-active polysaccharides from the natural resources.The researches about the polysaccharides from the fruit shell of Camellia oleifera Abel are not available.This paper is about the exaction, purification and antitumor activities of polysaccharides from the fruit shell of Camellia oleifera Abel.(1) Optimization of the extraction technologyUsing the fruit shell of Camellia oleifera Abel as raw material,the content of polysaccharides was determined by the method of phenol-sulphate acid.On the basis of single factors tests,the effects of the four factors including temperature,time,solid-liquid ratio and the concentration of ethanol on the yield of the polysacchafides of fruit shell of Camellia oleifera Abel were studied through orthogonal experiment design.The results showed that the temperature of extraction had " significant effect on the yield of polysaccharides.And the time of extraction,solid-liquid ratio,the concentration of ethanol had no significant effect on the yield of polysaccharides.The optimum technological parameters were as follows:temperature 90℃,extraction time 1h,solid-liquid ratio 1:15 and ethanol concentration at 80%.The yield of the polysaccharides was 5.93%under the optimum technological parameters.(2) Separation and purification researchThe protein was removed by Sevag method.After 7 times′operation,the polysaccharides removal rate of CPPS reached 49.8%,but the protein removal rate was only 16.8%.Sevag method was not effective in protein removing.Then taking the removal rate of polysaccharides,protein and polyphenolic compounds as the main value index,the most suitable kind of macroporous resin was selected out with static adsorption model.DA-201 was determined as the most suitable kind of macroporous resin.The static adsorption model results showed the protein removal rate of CPPS reached 55.0%and the polyphenolic compounds removal rate of CPPS reached 76.2%. DA-201 macroporous resin could remove the protein and polyphenolic compounds in CPPS effectively and it was better than Sevag method in protein removing.After the DA-201 macroporous resin separation,the two main fractions were obtained:CPPS1 and CPPS2.The polysaccharide content of CPPS1 was improved by 29.4%,the content of protein was decreased by 36.6%and the content of polyphenolic compounds was decreased by 52.3%compared with the composition of CPPS.Then CPPS1 and CPPS2 were further purified with DEAE-52 column and CPPS1-1,CPPS1-2,CPPS2-1 and CPPS2-2 were obtained(3) Composition analysisHigh performance liquid chromatography was used to analysis the distribution of molecular weight of polysaccharides and gas chromatography was used to analysis the monosaccharides. The HPLC results showed that the distribution range of molecular weight of CPPS was from 2.9×10³ to 2.8×10⁵.The gas chromatography results showed that CPPS was composed of six kinds of monosaccharides at least,which contained rhamnose,fucose,arabinose,mannose, glucose and galactose in the percentage of 6.65%,19.07%,2.67%,7.05%,48.76%and 15.78%. CPPS1-1 had the same monosaccharides types as the CPPS.The monosaccharides percentage of CPPS1-1 was different from CPPS.The monosaccharides of CPPS1-1 were in the percentage of 4.66%,8.84%,4.37%,8.86%,57.19%and 6.06%.Galactose was the main monosaccharide of the polysaccharides from the fruit shell of Camellia oleifera Abel.(4) Antioxidant activity researchTaking ascorbate as the control,the antioxidant activity of the different extracts from the fruit shell of Camellia oleifera Abel were determined by DPPH assay,FRAP assay,TEAC assay and Fenton Reaction,respectively.The results suggested that CPPS showed potent antioxidant activity. And the antioxidant activity of CPPS was concentration-related.The antioxidant capability of the three fractions and ascorbate decreased in the order of ascorbate＞water extract＞ethanol supernatant＞CPPS.(5) Atitumor activity researchThe inhibitory effects of CPPS on Hela,H460 and HepG2 were examined by MTT colorimetric assay in vitro,and the mouse tumor model was used to investigate the effects of polysaccharide on tumor growth in vivo.The results showed that the CPPS could inhibit proliferation of Hela,H460 and HepG2 and the inhibitory capability was concentrations-related. The inhibition rate reached 74.54%,51.38%and 63.42%for HepG2,Hela and H460 at the dose of 500μg/mL.CPPS also showed good inhibitory effect on the growth of S₁₈₀ sarcoma-transplanted mice,the inhibition rate of S₁₈₀ sarcoma reached 38.33%at the dose of 450 mg/kg.The results revealed that the polysaccharides of fruit shell of Camellia oleifera Abel was a kind of bio-active polysaccharides that has great research value and exploitation potential.