Study On Isolation And Purification、Structure Identification And Immunity Activity Of The Polysaccharides From Blueberry

Blueberry,also known as bilberry or blue berry, belonging to the Vaccinium genus of Ericaceae, is a kind of deciduous shrub plant. It has fascinated the whole world for its higher value in health care and it is recommended by FAO as one of the most healthy fruits. Blueberry contains a variety of active ingredients, such as phenolic acid, anthocyanin and flavone etc., which have been studied intensively both home and abroad. But detailed reports on the polysaccharides from Blueberry are not seen yet and there is information shows that blueberry residue contains many polysaccharide substances (Pan Yifeng,2005), which has great potential for development and utilization. This paper, taking Lanfeng breed as raw material, extracted, purified, separated and indentified the polysaccharides from blueberry and carried out a detailed study on its anti-tumor and immunoregulation role in vivo and some activities in vitro. This study has laid a theoretical basis for future in-depth research and utilization of blueberry polysaccharides and its study results are as follows:(1) Immersion method, enzymatic method and ultrasonic wave method were used separately in extracting polysaccharides from blueberry and the extraction process was optimized by orthogonal test to acquire the optimal process conditions of each method. Immersion method:ratio of material to water 1:60 g/mL,Temperature 90℃, time 4 hours, the yield of blueberry polysaccharide is 2.12%. Enzymatic method:enzymolysis time 100 mins, temperature 40℃, enzyme additive amount 0.6%, ratio of material to water 1:60 g/mL, and the yield of polysaccharide is 2.32%. Ultrasonic-assisted extraction method:extraction time 40mins, temperature 50℃, power of ultrasonic wave 80 W, ratio of material to water 1:60 g/mL, the yield of polysaccharide is 2.34%. The results indicated that the ultrasonic-assisted method is characterized by low cost, high efficiency and the more extractive quantities. Therefore, the ultrasonic-assisted method has already been suggested as a way of extractiving polysaccharides from blueberry.(2) Effect of de-protein was compared between Sevag method, TDA method and Enzyme method combined with Sevag, the results showed Enzyme method combined with Sevag has higher de-protein efficiency. Its optimal treatment conditions are:papain usage is 0.15%, pH 6.5,60℃constant temperature for 2 hours, supernatant was acquired by centrifugation, then de-protein 4 times by Sevag method to basically remove protein thoroughly. And this method showed better retention ratio for polysaccharide than the first two methods, blueberry polysaccharide retention ratio can reach 78.28%, indicating that it is a better method.(3) Hydrogen peroxide was used for decoloration. Considering the decloration rate and the retention rate of the final sample, the better choice is the hydrogen peroxide additive amount is 40%, pH 8.5, temperature 50℃, treatmeat time 4 h.The resultant decoloration rate and retention rate of blueberry polysaccharide are 93.11% and 79.24% respectively.(4) After decoloration and de-protein, blueberry polysaccharide went thorough DEAE-52 cellulose colum chromatography and then washed out by distilled water, 0.1mol/L NaCl、0.3 mol/L NaCl、0.5 mol/L NaCL in order, the four components of BBP1、BBP2、BBP3 and BBP4 were acquired at 11.77%、14.08%、34.65% and 10.35% yield respectively. BBP3 was further purified by going through Sephacryl S-300 chromatography and washing out by 0.1 mol/L NaCl to get BBP3-1 and BBP3-2 components. BBP3-1 was tested and verified as single polysaccharide through ultraviolet absorption spectrometry, Sephacryl S-300 chromatography and HPLC analysis.(5) Physicochemical properties research shows:BBP3-1 is white powder solid, no special odor, easy soluble in hot water and insoluble to organic solvents, like ethanol, ether and acetone etc. Its specific rotation in water solution is [α]20D=+17°. This polysaccharide was tested and verified as a kind of none-starch acid polysaccharide which does not contain N element, phenol, amino acids or protein. The weight-average molecular weight (Mw) of BBP3-1 is 18643 Da, the number-average molecular weight (Mn) of it is 9658 Da and the peak position molecular weigh (Mp) is 3554 Da and the distribution width of molecular weight (Mw/Mn) is 3.186. Viscosity character of blueberry polysaccharide research indicates: solution concentration, temperature, NaCl concentration, pH, heat treatment and shearing rate all have some certain effects on the viscosity of BBP.(6) The constituents of the monosaccharide BBP3-1 were determined by gas chromatography (GC) as:rhamnose, galactose and glucose at molar ratio of 1:1.5:2. Infrared spectrum(IR) displayed that the BBP3-1 has the characteristic absorption peak of polysaccharides and therefore, it was inferred that it is a kind of acid polysaccharide with uronic acid. It containsβ-D-glucopyranose or galactose, but does not contains mannose. Through nuclear magnetic resonance(NMR) analysis, it may infer that the BBP3-1 is aβ-polysaccharide of which the 1-6-glucopyranose chain as its main chain and it contains rhamnose, galactose and glucose.(7) S180 tumor-bearing mice model was established through lavage with 30d blueberry polysaccharide solution at 400 mg/Kg weight.d、200 mg/Kg weight.d、100 mg/Kg weight.d separately. The results indicated that the blueberry polysaccharide can significantly inhibit the mouse tumor’s growth and lift up the thymus index, spleen index of the tumor-bearing mice, notably enhance the proliferation capacity of the tumor-bearing mice’s spleen lymphocyte, markedly strengthen the cytophagy of the macrophage inside the tumor-bearing mice’s abdominal cavity on neutral red, strikingly encourage the secretion of TNF-α、IFN-γ、IL-2, sharply increase the viability of NK cell of the tumor-bearing mice, indicating that the blueberry polysaccharide can inhibit tumor’s growth through immune regulatory system. The lowest dosage of 100mg/Kg.d showed best effect. The result of delayed hypersensitivity induced by SRBC showed that blueberry polysaccharide in high dosage of 400 mg/Kg体重.dinhibits delayed hypersensitivity significantly.(8) Drug delivery of blueberry polysaccharide in vitro can also significantly boost the immunologic function of the normal mice. The study showed that blueberry polysaccharide significantly promotes the transformation capacity of the spleen lymphocyte, but there exists a mutual inhibitory action between it and ConA. Blueberry polysaccharide can also significantly strengthen the cytophagy of the macrophage on neutral red, also there exists a significant synergy between it and ConA.(9) Some functional properties of blueberry polysaccharide were studied. Anti-oxidation test in vitro showed the blueberry polysaccharide has strong clearing effect on OH·and DPPH·radicals and its clearing rate increases with its concentration increasing, but it has hardly any effect on O2·-. The test of blueberry polysaccharide on soybean oil and lard oil’s anti-oxidation showed it has some effect on anti-lipid peroxidation, but its effect is not significant. It also found it has synergy effect with citric acid. By using blueberry polysaccharide’s clearing action in vitro on a-amylase, the effect of lowering blood-glucose was evaluated. The test showed that it has some certain effect on clearingα-amylase. Through the inhibitory effect test on several common strains, it showed that blueberry polysaccharide do not have significant inhibitory effect.