| Litchi is delicious and nutritiou, is saw as "the treasure of the fruit", all parts of which could be used as medicine. Studies have shown that litchi could improve antioxidant,antitumor and immunomodulatory effect and clearly pointed out that litchi polysaccharides (LCP) is one of the main functional active components. In this paper, litchi pulp was used as raw materials, the optimum conditions of ultrasonic-microwave-enzyme -assisted extraction technology for LCP were determined; column chromatography on DEAE-52 cellulose was used to isolate and purify the major polysaccharides extracted from litchi pulp; the antioxidant,immunomodulatory effects and strucure of crude LCP and major purify LCP were compared. The study results will offer a new clue for the deep processing of LCP, as well as provide theoretical foundation and technical guidance for the development of healthy products. The main contents and results are shown as below:1. Optimization of extraction condition of LCP:The process of using ultrasonic-microwave-enzyme synergistic method to extract polysaccharides from litchi pulp was optimized through the response surface methodology of Box-Benhnek central combination experimental design. The regression model of five factors for the extraction of litchi polysaccharides, which were the amount of enzyme addition, solid-liquid ration, temperature and time was established. Through the experiments and analysis, the optimal process conditions to extract litchi polysaccharides were as follows:the amount that cellulose added wasl4mg/g (enzyme activity was 30U/mg), the solid-liquid ratio was 1: 10, the temperature was set at 49℃, the pH value was 4.8 and the time used wasl2min. From those conditions, the yield rate of polysaccharides extraction was 23.3%, which were 19.0%,4.4% and 17.1% (P<0.05) higher than those were extracted using the method of traditional hot water, ultrasonic and microwave respectively.2. Comparison of functional activity and structure of LCP from ultrasonic-microwave- enzyme -assisted extraction technology and hot water:The antioxidant activity of extract from Litchi was assayed by ferric reducing (FRAP) assay, DPPH and ABTS radical scavenging and the reslutes from ultrasonic- microwave-enzyme -assisted extraction technology showed that FRAP value of LCP from ultrasonic- microwave-enzyme -assisted extraction technology was 0.32μmol FeSO4/mg, higher than hot water(0.16μmol FeSO4/mg), the positive correlation (P<0.01) existed between reductive capacity and concentration(P<0.01); IC50 value of DPPH and ABTS radical scavenging activity was respectively 427μg/mL,203μg/mL, lower than hot water (DPPH and ABTS radical scavenging activity was respectively 2734μg/mL and 1590μg/mL). At the same time, the effect from ultrasonic -microwave -enzyme -assisted extraction technology was better than hot water to increase the cytotoxic activity of NK cells(P<0.05) and promote ConA-induced proliferation of the spleen lymphocyte (P<0.01).Analysis results of physiochemical properties showed that the total sugar content of LCP from ultrasonic -microwave -enzyme -assisted extraction technology was 57.26%, the aldonic acid content was 12.39%; the total sugar content of LCP from hot water was 47.62%, the aldonic acid content was 23.69%. Analysis results of HPGPC,GC-MS,IR and UV showed that LCP from ultrasonic -microwave -enzyme -assisted extraction technology was polysaccharides includingα-pyrans and haligen, composed of xylose,rhamnose,ribise,mannose,glucose,galactose,arabinose, and the molar ratio was 1: 4.39:6.05:16.07:65.20:23.62:15.25, its relative molecular weight was 7.92×104,9.09×103; LCP from hot water was protein-polysaccharides with O-glucosides and protein, composed of xylose,rhamnose,ribise,mannose,glucose,galactose,arabinose, and the molar ratio was 1:2.59:5.77:8.13:22.49:22.50:26.02, its relative molecular weight was 6.01×104.3.Isolation and purification:The DEAE-52 ionic exchange chromatographic method was appied to purify the rude polysaccharide, and five components obtained and the ratio was 91.10%:0.78%:0.42%:0.11%:1.09%.The optional isolation conditions were established as:the column loading:4.2mg/mL; the column volume:8mL; the elution speed:1mL/min; the ratio of diameter and length:30:1; operate twice.4.The analysis of active components of LCP:The antioxidant activity of LCP1 and LCP5 was studied by FRAP, and the immunomodulatory effect was evaluated by using normal mice immunocytes with respect to lymphocytes proliferation and NK activity. The results indicated that the FRAP of LCP5(1.281μmol/mg) was higher than LCP1(0.136μmol/mg, P<0.05). LCP5 was better than LCP1(P<0.05) to increase the cytotoxic activity of NK cells and at the dose of 300～400μg/mL, LCP5 was better than LCP1(P<0.05) to promote ConA- induced proliferation of the spleen lymphocyte (P<0.05).Analysis results of physiochemical properties showed that the total sugar content of LCP1 was 63.16%, the aldonic acid content was 32.36%; the total sugar content of LCP5 was42.84%, the aldonic acid content was 10.82%. Analysis results of HPGPC,GC-MS,IR and UV showed that LCP was acid polysaccharides with acetylene series, composed of ribise,rhamnose,mannose,glucose,arabinose,galactose and the molar ratio was 1: 1.58:2.66:4.97:9.15:11.14, its relative molecular weight was 5.45×104; LCP5 was acid polysaccharides with halogen, composed of ribise,rhamnose,mannose,glucose, arabinose,galactose and the molar ratio was 1:0.75:1.49:3.20:4.82:2.09, its relative molecular weight was 1.41×104. |
PDF Full Text Request