[0015] The implementation of the present invention will be described in detail below with reference to specific embodiments, so as to fully understand how the present invention applies technical means to solve technical problems and achieve the realization process of technical effects and implement them accordingly.
[0016] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and products used are also commercially available. The source of the reagents used, the trade name, and those where it is necessary to list their constituents are all indicated when they first appear.
[0017] The embodiment of the present invention is:
[0018] A method for extracting Sargassum polysaccharides includes the following processing steps:
[0019] a. Constant temperature water bath treatment: Sargassum and dilute acid solution (adjust pH=2 with hydrochloric acid solution) with a material-to-liquid ratio of 1:120, in a constant temperature water bath at 97°C for 210 minutes to remove impurities to obtain an extract, which is used 4mol/L NaOH is neutralized to pH 7.0;
[0020] b. Pretreatment of the extract: The extract is concentrated by a rotary evaporator at 70°C. After the concentrated solution is cooled to room temperature, add 4 times the volume of anhydrous ethanol to alcohol precipitation, place in a refrigerator at 4°C overnight, centrifuge to discard the supernatant, and add distilled water to dissolve the precipitate. Repeated freezing and thawing of the obtained solution, centrifugation to remove impurities (3500 rpm, 10 min), freezing conditions at -20°C, dissolution conditions at 50°C, repeating 7 times, and then drying with a freeze dryer to obtain crude product 1;
[0021] c. Crude product 1 deproteinization: Crude polysaccharides are dissolved in distilled water at 1:25 to obtain sugar solution, then add sevage reagent (chloroform:n-butanol=4:1) of the total volume of sugar solution, and shake for 3h. Centrifuge (4000 rpm, 5 min) to take the upper sugar solution, repeat 12 times; freeze and thaw the sugar solution, centrifuge to remove impurities (3500 rpm, 10 min), freezing condition is -20 ℃, dissolving condition is 50 ℃, repeat 5 times, then Dry by freeze dryer to obtain crude product 2;
[0022] d. The crude product is separated and purified by column chromatography 2:
[0023] (1) Pretreatment of DEAE-Sepharose CL-6B gel: Put the gel in a vacuum dryer, connect to a circulating water vacuum pump, perform intermittent vacuuming, pump air for 30 minutes, rest for 5 minutes, and stir with a glass rod to make It is loose, so that all the gas in the gel can be extracted; if there is gas in the gel, it will affect the separation and purification of polysaccharide samples;
[0024] (2) Column loading and balance: Clamp the column on the iron stand and keep it vertical: pour the balance buffer (distilled water) from the top of the column to remove the dead corners and the air in the outlet pipe. After the bubbles are removed, make distilled water Keep a height of 3cm in the column and close the column outlet; use a glass rod to gently stir the gel suspension, introduce the gel suspension into the column through the glass rod, and add the gel suspension from the column entrance along the inner wall of the column to avoid bubbles Column diameter: 4.6cm, height: 40cm; When the gel settles, turn on the constant flow pump, adjust the flow rate to about 60mL/h, balance with about 2000mL of distilled water to make it compact and firm;
[0025] (3) Sample loading detection and collection: Dissolve 500 mg of crude product 2 in 10 mL of distilled water, aspirate the upper layer of the column buffer to be level with the surface of the gel, draw the sample solution and slowly add it in the same direction as the inner wall of the chromatography column, so that the sample is on the gel column The surface is evenly distributed; after adding, turn on the constant flow pump to make the sample liquid enter the column. When about 1mm of sample liquid remains on the upper surface of the column, add distilled water slowly along the inner wall of the column by rotating it to make the wall stick After washing with liquid, continue to add distilled water to a height of about 8cm from the upper surface of the gel column according to this method; cover the column; adjust the flow rate to 60ml/h, elution with distilled water and 1mol/L NaCl linear gradient respectively, set 12min Collect one tube, each tube is 12ml, automatic partial collector collects 100 tubes;
[0026] Use the phenol-sulfuric acid method to determine the sugar content distribution, use the ultraviolet spectrophotometry to determine the protein content distribution, record the data and make the elution distribution map, collect the eluate according to the peak range; use a dialysis bag with a molecular weight cut-off of 3500 to collect the liquid Tap water flowing water was dialyzed for 24 hours, and then distilled water was dialyzed for 24 hours. After the dialysis was completed, it was concentrated by rotary evaporation and freeze-dried to obtain the Sargassum polysaccharide.
[0027] The homogeneity of the freeze-dried samples was detected by Agilent1100 system, and the conditions were: liquid chromatography column ShodexSugarKS-804, flow rate 1mL/min, temperature 50℃, differential detector.
[0028] After collecting the NaCl eluent, it is necessary to regenerate the column, wash the column with 200ml of 1M sodium hydroxide, and then wash the column with 2000ml of distilled water before reloading the sample.
[0029] The method for extracting Sargassum polysaccharides of the present invention includes mixing Sargassum and dilute acid in a constant temperature water bath to obtain an extract, pretreating the extract to obtain crude product 1, and then deproteinizing crude product 1 to obtain crude product 2. Separation and purification, the above-mentioned method is not time-consuming, energy-saving, and the yield of polysaccharides is high. The extracted polysaccharides have strong antioxidant activity and cancer cell growth inhibitory effect. Detected by high performance liquid chromatography, the water eluted component got a homogeneous component (named STPⅠ), and 1mol/L NaCl linear gradient elution got a homogeneous component (named STPⅡ). Both STPⅠ and STPⅡ have strong antioxidant activity and cancer cell growth inhibitory effect; STPⅠ has an IC50 value of 1.00 mg/mL for scavenging superoxide free radicals and an IC50 value of 0.145 mg/mL for hydroxyl free radicals. The growth inhibitory effect of caco-2 has an IC50 value of 2.554 mg/mL; the IC50 value of STP Ⅱ for scavenging superoxide free radicals is 0.131 mg/mL, and the IC50 value for scavenging hydroxyl free radicals is 0.106 mg/mL, which is against colon cancer cells caco-2 The IC50 value of the growth inhibitory effect is 4.071mg/mL.
