1 preface

Polysaccharide is a polymer in which aldose and/or ketose exist in nature and are linked together by glycosidic bonds. Polysaccharide is an essential component of all living things, which is closely related to various physiological functions of maintaining life. In recent years, polysaccharides from plants, marine organisms and fungi have become an important class of natural products with biological activities. Many polysaccharides have been found to have anti-tumor, immune, anticoagulant, hypoglycemic and antiviral activities. Polysaccharide has a complex structure, because it has many different monosaccharide residues, different connection positions and different types of glycosidic bonds, and can form secondary structures with different conformations, different relative molecular weights and hydrogen bonds within and between chains.

The research on polysaccharide began in 1940s, but it attracted great attention as a broad-spectrum immune enhancer in 1960s. After more than 40 years of continuous development, people have a new understanding of polysaccharide, an important life substance, making this subject one of the most active research fields in life science [1]. More and more studies have found that polysaccharides have great utilization value to human body, which can be divided into three categories according to their sources: animal polysaccharides, plant polysaccharides and microbial polysaccharides. Among them, ginseng, scutellaria baicalensis, acanthopanax senticosus, aloe and other plant polysaccharides have obvious medicinal effects, such as enhancing immunity, anti-tumor and anti-radiation. According to literature [2], nearly 100 kinds of plant polysaccharides have been isolated and extracted. This kind of polysaccharide has a wide range of sources and no cytotoxicity, and has little toxic side effects when applied to organisms, so the research on plant polysaccharides has become a hot spot in the medical field.

Polysaccharide (PS) is also called polysaccharide. It exists in animals, higher plants, microorganisms (bacteria and fungi) and algae. Polysaccharide has complex and multifaceted biological activities and functions, and can be used as a broad-spectrum immune promoter with immunomodulatory function. For example, polysaccharides can treat immune system diseases such as rheumatism, chronic viral hepatitis and cancer, and even resist HIV [3]; It also has the functions of anti-infection, anti-radiation, anticoagulation, blood sugar reduction and blood lipid reduction; Promote the biosynthesis of nucleic acids and protein; It can control cell division and differentiation, regulate cell growth and aging, and polysaccharide as a drug has little toxicity, so the research on polysaccharide has aroused great interest. The molecular weight of polysaccharide is tens of thousands or millions. Plant polysaccharides include starch, cellulose, dextran (such as lentinan), fructan gum, mucus, viscose (such as agar) and so on. Some of them are cancer cell inhibitors, but their specificity is poor. In 1980s, Franz G and other research groups in Germany were the most active, which proved that many plant polysaccharides and fungal polysaccharides had anti-tumor activity. These include lentinan, Ps-K, Coriolus versicolor, Polyporus umbellatus, etc., which have been applied in clinic.

Polysaccharide is also a good adjuvant in medicine. In recent years, it has been found that the sugar chain of polysaccharide plays a decisive role in molecular biology. In addition, it can control cell division and differentiation, and regulate cell growth and aging. Polysaccharides are also widely used in food industry, fermentation industry and petroleum industry. More and more plant polysaccharides have been newly developed, and their application value in food field has been developed and confirmed, which has injected fresh vitality into the rapid development of food industry. In recent years, with people's understanding of the role of polysaccharides and glycoconjugates in life science and the rapid development and improvement of polysaccharide research technology, polysaccharide research has sprung up like mushrooms after rain. The international scientific community regards polysaccharide research as the frontier field of life science, and even puts forward that 2 1 century is the century of polysaccharides [4]. Many plant polysaccharides have immunomodulatory activity, which can enhance the functions of immune systems such as macrophages and lymphocytes, activate or promote the production of antibodies and complements, and induce interferon. Polysaccharide can also be used to prevent and treat diabetes, promote the healing and repair of gastric ulcer, and has many functions such as radiation resistance and blood lipid reduction. Therefore, polysaccharides and their derivatives, as biological effect regulators, have become one of the research hotspots of natural drugs in recent years.

Therefore, it is of great practical significance to study the separation and purification of polysaccharides. This paper mainly studies alcohol precipitation classification, column chromatography and so on.

2 experimental part

2. 1 main reagents and instruments

Dextran DEAE series, Pharmacia company

Salivary amylase, Sigma Company, USA

SDS gel

Coomassie blue reagent

β -glucosyl derivative reagent

Other reagents are analytically pure.

PD 10 column, amersham harmcia biotechnology company.

Automatic flow separation collector

RE52CS rotary evaporator, Shanghai Medical Machinery Special Machine Factory.

60 10 ultraviolet-visible spectrophotometer, Angel Shanghai Analytical Instrument Company.

American Waters Company Model 2690 High Performance Liquid Chromatograph

2.2 experimental method office "/>

2.2. 1 starch removal

20% water solution was prepared from crude polysaccharide of Ornithogalum caudatum, and starch was removed by salivary amylase at 37℃.

Protein removal

Protein was removed by Sevage method: chloroform and n-butanol were added to the sample aqueous solution according to the volume ratio of 4: 1 (the ratio was 5: 1), and the gel-like protein was removed by centrifugation (10000rpm, 20min), and repeated several times (3 times) until protein was completely removed. Ninhydrin reaction was used to detect the protein removal effect, and the result was negative (or biuret test); At the same time, the ultraviolet absorption curve of deproteinized samples was measured at 200~280 nm to test the effect. After deproteinization, the UV absorption peak of polysaccharide solution disappeared at 260 ~ 280 nm, indicating that protein was not contained in polysaccharide.

desalt

Using a semipermeable membrane, the above-mentioned treatment solution was dialyzed with reverse running water to remove small molecular impurities such as oligosaccharides, and then dialyzed with distilled water for 48 hours.

Classification of alcohol precipitation

Dissolve crude polysaccharide 15g+250mlH2O by heating, cooling, adding 40% ethanol for precipitation, stirring, standing, filtering to obtain precipitate (OC- 1), precipitating with 40% ethanol for three times, combining filtrates, and concentrating.

Precipitating the concentrated solution with 60% ethanol, stirring and dissolving, standing and filtering to obtain precipitate (OC-2). The filtrate was precipitated with 60% ethanol for 3 times, and the precipitates were combined. The filtrate (60% ethanol solution) (OC-3) was obtained.

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Published on February 9, 2009 14: 17 | published by the author only.

2.2.5 column separation traffic "/>

60% ethanol precipitate (OC-2) was dissolved in hot water and mixed with dextran gel DEAE resin in a beaker. After equilibrium, the resin was placed in a separatory funnel and washed with 0.5 mol/L NH4CO3 solution, which was divided into two components: the eluent of 0.5 mol/L NH4CO3 solution (OC-2- 1) and the component combined with the resin (OC-2-2).

Evaporating the eluent (OC-2- 1) of 0.5mol/L ammonium bicarbonate solution, dissolving it in water, loading it on DEAE column (4.5× 12.0), and then eluting it with 0.2mol/L ammonium bicarbonate solution, and determining the OD value of each eluent. Draw elution curve according to the number of collecting tubes. Components with the same peak are combined to produce seven components: OC-2- 1-a (unbound fraction), OC-2- 1-b, OC-2- 1-c, OC-2- 1-d and OC-2.

The absorbance OD values of the above different components were measured by ultraviolet spectrophotometer at the wavelengths of 2 15 and 280nm.

The components were evaporated to dryness under reduced pressure below 60℃, and then the salts and small molecules were removed by PD 10 column (Sephadex G-25M).

Precipitate and dissolve some hot water with 60% ethanol on DEAE resin column, and use different concentrations of ammonium bicarbonate: 0.2M, 0.25M, 0.4M, 0.5M,1.0m.