| Objective:To purification and molecular weight of Lan Qi compound polysaccharide in Isatidis Radix, Astragali Radix and Epimedium.Method and Result: Based on single-factor experiment and using orthogonal experimental design to optimize the best technology of Isatidis Radix, Astragali Radix,Epimedium polysaccharide.The best extractive condition of Isatidis Radix was:distilled water, ratio of water to material was 15, was soaked for 30 minutes under room temperature, and digeratur extraction for 3 hours under 90℃, extracted 2 times.The extracted liquid were pooled and vacuum concentrated to 1 times the amount of material, centrifugated by 5000 rpm for 10 minutes, and liquid supernatant was joined into ethanol, still standed 12 hours with 75% ethanol, the extraction rate of Isatidis Radix polysaccharides was 2.34%. The method of Sevage is used to remove protein.By D152 cation exchange resin to optimize the best decoloring process: D152,adsorption time 180 min, the sample one column volume, elution velocity control 2ml/min, the rate of polysaccharide retention,protein removal and decolorization are93.91%, 77.19% and 61.51%.Using dialysis bag of 3000 intercept molecular weight to remove small molecules in 48 hours.with different concentrations of salt by DEAE Sephadex A-50 resulting from the elution A, B, C, D, E five components and using Sephadex G100 to get five polysaccharide sample liquid. Ultraviolet spectrometry to prove the polysaccharide sample liquid have no protein and nucleic acid in 280 nm and 260 nm. Shodex OHpak SB-804 HQ 8.0mm×300mm column, the eluent is 100%super purified water, Detector: 380-ELSD evaporative light detector, the standard linear relation of dextran is lg Mr=-1.976RT+19.88,(R2=0.9941), linear range is in2.0E+04~5.0E+05. The result shows that: Isatidis Radix polysaccharides A contains its molecular weight is3.2E+05,1.9E+05,1.1E+05,B contains its molecular weight is2.8E+05and1.8E+05, C contains its molecular weight is 3.1E+05, 1.7E+05, 3.9E+04,D contains its molecular weight is 9.5E+03, E contains its molecular weight is1.3E+03.The best extractive condition of Astragali Radix was: distilled water, ratio of water to material was 8, was soaked for 30 minutes under room temperature, and digeratur extraction for 1 hours under 90℃, extracted 3 times. The extracted liquid were pooled and vacuum concentrated to 5 times the amount of material,centrifugated by 5000 rpm for 10 minutes, and liquid supernatant was joined into ethanol, still standed 12 hours with 70% ethanol, the extraction rate of Astragali Radix polysaccharides was 2.97%. The method of Sevage is used to remove protein. By D152 cation exchange resin to optimize the best decoloring process: D152, adsorption time 180 min, the sample concentration is 0.08 g/m L,one column volume, elution velocity control 1 ml/min, the rate of polysaccharide retention,protein removal and decolorization are 96.94%,62.67% and 54.96%.Using dialysis bag of 7000 intercept molecular weight to remove small molecules in 24 hours.with different concentrations of salt by DEAE Sephadex A-50 resulting from the elution A, B two components and using Sephadex G100 to get three polysaccharide sample liquid.Ultraviolet spectrometry to prove the polysaccharide sample liquid have no protein and nucleic acid in 280 nm and 260 nm. With the same method to determine the molecular weight of Astragali Radix. The result shows that: Astragali Radix polysaccharides A1 contains its molecular weight is 1.2E+04, A2 contains its molecular weight is1.6E+05and 7.5E+04,B contains its molecular weight is 3.4E+04.The best extractive condition of Epimedii Folium was: distilled water, ratio of water to material was 20, was soaked for 30 minutes under room temperature, and digeratur extraction for 2 hours under 90℃, extracted 2 times. The extracted liquid were pooled and vacuum concentrated to 1 times the amount of material,centrifugated by 5000 rpm for 10 minutes, and liquid supernatant was joined into ethanol, still standed 12 hours with 70% ethanol, the extraction rate of Epimedii Folium polysaccharides was 2.04%. The method of Sevage is used to remove protein.By ADS-7 cation exchange resin to optimize the best decoloring process: ADS-7,adsorption time 180 min, the sample concentration is 0.33 g/m L,1.5 times column volume, elution velocity control 1 ml/min, the rate of polysaccharide retention,proteinremoval and decolorization are 88.42%,54.07% and 63.59%.Using dialysis bag of3000 intercept molecular weight to remove small molecules in 24 hours.with different concentrations of salt by DEAE Sephadex A-50 resulting from the elution A, B,C three components and using Sephadex G100 to get three polysaccharide sample liquid.Ultraviolet spectrometry to prove the polysaccharide sample liquid have no protein and nucleic acid in 280 nm and 260 nm. With the same method to determine the molecular weight of Epimedii Folium. The result shows that: Epimedii Folium polysaccharides A contains its molecular weight is 1.3E+05, B contains its molecular weight is 3.5E+05,C contains its molecular weight is 6.2E+03.Conclusion: Research result shows, the extractive,purified and molecular weight technique are rational and feasible, can be use to refine and determine molecular weight of Lan Qi Compound Polysaccharides. |
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