Study on the Preparation of the Low Molecular Weight Fuciodan from Sargassum Henslowianum (C.Agardh) and Anti-tumor Activity
|School||Guangdong Ocean University|
|Course||Agricultural Products Processing and Storage|
|Keywords||Sargassum henslowianum C.Agardh Classification and purification Degrading enzyme Low molecular weight fucoidan(LMWF) Enzymatic degradation Culture in vitro Anti-tumor effect|
Sargassum henslowianum C. Agardh is Phaeophyta membership, marjoram algae present, the Sargasso Branch, Sargassum spp. Fucadan is a active seaweed polysaccharide with sulfate groups which has significant anti-viral, antitumor, antibacterial, anticoagulant, and improve immunity and so on. However, because of its large molecular weight and bad solubility, the active polysaccharide are not easily absorbed,so its research and application are restrained. Enzyme degradation is a mild method, making low molecular weight seaweed polysaccharides, but the price of enzyme preparation is high and the effect of degradation is not good, so, looking for the more economical enzyme preparation deserves to further study. In this paper, taking the heinz Sargasso of NaoZhou island in Zhanjiang as raw materials, polysaccharide from Sargassum Heinz （SF） that had been initially purified were graded and then purified again. The enzymes will be extracted from liver and then pured, using pancreas of fresh ocean small miscellaneous fish as the material. And the high molecular weight polysaccharides of Sargassum Heinz will be degradated by enzyme, meanwhile the relationship between anti-tumor effect and the molecular weight will also be researched and analysed.Firstly, using eight kinds of liver, pancreas of fresh ocean small miscellaneous fish extracts a higher activity enzyme, the enzyme activity from the sand drill fish was higher than others. Taking phosphate buffer as extract, the pH, extraction time and extraction temperature were Optimized by single factor experiment and orthogonal test of L9（34）, the best extraction conditions were pH 6.0, extraction temperature 35℃, extraction time 3 h. Crude enzyme was purified by salting out step by step approach, part impurities were removed with 30% ammonium sulfate. Salting conditions （ammonium sulfate saturation, salting time, salting pH, salting temperature） were also optimized. The best conditions for salting is 80% ammonium sulfate, pH5.5, salt temperature is 45℃, salting time is 3 h. In addition, the enzyme was eluted out D1, D2 and D3 three components by dextran G-100 column chromatography. The enzyme activity of D2 is largest, D1, D3 have almost no enzyme activity, So the fractions of D2 will be collected and then freeze-dryed, saved at low temperature. Heinz Sargassum polysaccharides has been purified by anion exchange resin which were graded again by agar gel SepharoseCL-6B, getting four polysaccharide fractions: SF1, SF2, SF3, SF4. The molecular weight of four polysaccharides components were relatively uniform and the average molecular weights were: 851.13kDa, 239.85kDa, 102.34kDa, 19.95kDa by standard curve with known molecular weight standard glucan dextran. Four high molecular weight polysaccharides were degraded by enzyme, the degradation conditions: substrate concentration, degradation pH, temperature and time were optimized. With four factors and three levels （L9（34）） orthogonal experiment, the best degradation conditions were substrate concentration of 0.6%, pH5.0, temperature 45℃, the degradation time 4 h. After degradation, the molecular weight of four degradation components SF1-R、SF2-R、SF3-R、SF4-R were 53.74kDa, 19.54kDa, 9.27kDa, 6.69kDa by the best enzyme degradation conditions. Degradation times were 15.82, 12.34, 11.22, 3.01by comparing the molecular weight before and after degradation. By chemical analysis, the total sugar contents of SF1, SF1-R, SF2, SF2-R, SF3, SF3-R, SF4, SF4-R were: 72.92%, 70.15%, 67.55%, 65.46%, 60.87%, 59.23%, 56.81% ,55.78% by measuring; The contents of L-fucose were: 38.82%, 36.91%, 34.12%, 31.33%, 30.19%, 25.58%, 26.86%,19.84%; Sulfate contents were: 4.14%, 4.65%, 6.37%, 7.43%, 8.35%, 8.93%, 9.72%, 10.23%; Uronic acid contents were: 30.93%, 33.63%, 32.65%, 35.87%, 29.88%, 30.44%, 21.61%, 20.85%.The vitro anti-tumor effect of 8 components Sargassum polysaccharide for Mice sarcoma cells S-180, people lung cancer cells A549, people hepatumor cells SMMC-7721 which were at five different concentrations（1mg/mL,2.5 mg/mL,5 mg/mL,10 mg/mL,20 mg/mL ）on 24 h, 48 h, 72 h was determined by culturing cells in vitro.The results showed that the inhibition effect of 8 components Sargassum polysaccharide on 3 tumor cells were different at different concentrations and different time, with the Sargassum polysaccharide concentration increasing and time extensing, the inhibition effect of 3 tumor cells growing, showing the dependent of dose and time. By analysising statistically（P<0.05 significant,P<0.01 very significant ）, in 48 h72 h group, the inbibitional effect of SF1-R, SF2-R were more significant than SF1, SF2 on three tumor cells（P<0.05 significant,P<0.01 very significant ）; In 72 h group, the inbibitional effect of SF3-R was slightly higher than the inbibitional effect of before degradation（P<0.05 significant,P<0.01 very significant ）; In 24 h72 h group, the inbibitional effect of SF4-R was not significant （P>0.05）. Inhibition of three tumor cells showed : the inbibitional effect of SF2-R on the S-180, A549 were the best, the inhibition ratio respectively was 77.57%, 61.06% after 72 h; the inbibitional effect of SF1-R on SMMC-7721 was the best, the Inhibition rate reached 62.47% after 72 h. By calculating, the IC50 values of SF1, SF1-R, SF2, SF2-R, SF3, SF3-R, SF4, SF4-R on the S-180 after 72 h were: 7.11mg/mL, 5.57mg/mL, 6.61mg/mL, 4.89mg/mL, 9.91 mg/mL, 8.91mg/mL, 14.66mg/mL, 14.79mg/mL; The IC50 values on the A549 after 72h were:11.64mg/mL,9.86mg/mL,10.63mg/mL,8.75mg/mL,14.37mg/mL,12.62mg/mL,18.71mg/mL, 20.27mg/mL; The IC50 values on the A549 cells after 72 h were: 9.33mg/mL, 7.88mg/mL,11.48mg/mL,9.75mg/mL,13.66mg/mL,12.84mg/mL,15.94mg/mL, 16.83mg/L.