Study On The Extraction, Purification And Anti-tumor In Vitro Of Polysaccharides In Leaves Of Vaccinium Uliginosum L.

For the past years, Vaccinium uliginosum L., as a kind of food or medicine, has been considered as one of the five healthy foods by Food and Agriculture Organization, is drawing more and more attention in the field of food and health care products industries. Many researchers have been studying on flavonoids and anthocyanins in Vaccinium uliginosum L. systematically and thoroughly, and substantial results have been achieved, however, the research on the active polysaccharide from Vaccinium uliginosum L. has rarely been reported. Polysaccharide extracted from many plants have several physiological functions such as anti-tumor, anti-infection, anti-radiation, depressing blood glucose and blood fat and so on, meanwhile, it has been attracting more and more attentions from the researchers. In order to develop the resources of Changbai Mountain thoroughly, the polysaccharides in the Vaccinium uliginosum L. was used as the study object in the present paper.In this paper, common nutritional ingredients in trunk,leaves and fruits of Vaccinium uliginosum L have been studied, then phenol.sulphuric acid method was employed to assay the polysaccharide compounds and the standard curve of glucose was drew. The regression equation of standard curve was: y=0.68696 x+ 0.01111, with a high linear correlation (coefficient correlation is 0.99902) which indicated that the contents of polysaccharide could be determined accurately. And then the contents of polysaccharide in different part of Vaccinium uliginosum L. were determined, and the contents in different part of Vaccinium uliginosum L. were that: 10.33% in leaves, and 7.81% in trunk, 6.83% in frozen fruits as the lowest one.The extraction technology was studied systematically, the comparison of the thermo.refluxing method and advanced technology with respect to microwave,ultrasonic wave and supersonic technology assist water immersion method was conducted in the article, the optimized technology parameters were studied, respectively. It was found that the extraction rate of polysaccharide by thermo.refluxing method was the highest, reaching 11.03%. The moderate method was supersonic technology assist water immersion, of which the extraction rate reached 7.96% and the lowest one was ultrasonic wave, of which the extraction rate was 6.80%. By contrast, the thermo.refluxing method was the most convenient to carry out, especially with a low cost.The methods, including Sevage method and TCA method used to remove protein from rude Vaccinium uliginosum L. polysaccharides were employed to purify polysaccharides after the extraction process under the optimized extracting technology. The Sevage method was repeated 3 times during the experiment, the protein detaching rate was 78.61%, the reservation rate of polysaccharide was 82.05%; while in the TCA method, the protein detaching rate was 82.73%, and the retain rate of polysaccharide was 34.41%. By contrast,Sevage method was better than TCA method used to remove protein from rude Vaccinium uliginosum L. polysaccharides.According to an analysis of the effect of five macroporous resins in the aspect of decolorization effect, the results indicated that: AB-8 macroporous resin in the aspect of decolorization was better. Therefore, the AB-8 was selected. After the study on adsorption dynamics, AB-8 macroporous resin could satisfy Freundlich equation and Boyd liquid membrane diffusivity theory in the aspect of decolorization effect. The optimized conditions of decolorization were that, temperature 35℃, decolorization time 8h, macroporous resin amount(m/v)4:10, decoloration rate 65.31% on average, saccharide reservation rate reached 65.59% on average.The decolorization temperature, time and the amount of absorbite were optimized according to the mathematic model built by CentralComposite Design using Design ExpertSoftware on the principle of response surface methodology. The optimum regression equation was Y=66.41+0.78X1+3.38X2+7.48X3+0.57X1X2 +2.92X1X3 +1.97X2X3 +1.41X12-2.48X22 -2.58X32. The best technology conditions was that: temperature 35.4℃,time 79.3min, usage amount of absorbite 1.45%, decoloration rate could get to 70.97%.Leaves of Vaccinium uliginosum L. polysaccharides(LVP) after protein removal, decoloration and dialysis was further purified by Sephadex G.75 twice, and Sephadex G.25 once, eluting reagent were deionized water, 0.1mol/L NaCl and deionized water in order, LVPⅣwas obtained by further purification.The specific rotatory power of LVPⅣwas [α]D20+11.5. While the structure of LVPⅣwas chiefly combined withα-glycoside bonds. LVPⅣwas a kind of white powder, it can be dissolved by water, especially hot water, it can not be dissolved by methanol, ethanol, acetone, acetidin and n.butanol etc. After evaluation by qualitative reaction, protein, nucleinic acid, starch, reducing sugar couldn't be detected in LVPⅣ. After LVPⅣhave been hydrolyzed completely, its composition of monosaccharide was determined by gas chromatography. The unit monosaccharide of LVPⅣwas identified as glucose, galactose and two unknown monosaccharides with a ratio of 3.16:1:1.73:2.49. The new polysaccharides LVPⅣwas isolated and purified from leaves of Vaccinium uliginosum L. polysaccharides (LVP). Two unknown monosaccharides must be determined by GC-MS further.Total sugar was determined by phenol . sulphuric acid method and aldonic acid was determined by sulphuric acid.carbazole method, the pure degree of water soluble polysaccharide in LVPⅣwas 99.10%, among the total ,aldonic acid was14.46%, neutral saccharide was 84.64%.In the end, the antineoplasmic activity of polysaccharide samples on S180 cell was studied by MTT bioassay in vitro.The results showed that all the samples had apparent antineoplasmic activity on S180 cell, the antitumor effects will strengthen with the increasing concentration in LVPⅣ. Through a comparative analysis of different times on the effect of antineoplasmic activity on S180 cell, the result indicated that diffierent times on the effect of antineoplasmic activity on S180 cell was not significant.A systematical research on the extraction and purification of leaves of polysaccharides from Vaccinium uliginosum L. was studied in the laboratory; however, the semi.works production remained to be solved in the future. The antineoplasmic activity in this experiment presented herein was only a part of the functional activities of polysaccharides, yet the other activities such as antioxidation, anticoagulated blood, antiradiation, hypoglycemic and hgpolipidemic activity were needed to be carried out in future.