Investigating Polysaccharides: Methods and Protocols

Polysaccharides are made by polymerizing more than 10 monosaccharide molecules through glycosidic bonds. The molecular weight of polysaccharides is relatively large, usually consisting of hundreds or even tens of thousands of monosaccharide molecules. Polysaccharides, together with nucleic acids, proteins, and lipids, are called the four basic substances of life and play important roles in many life activities. The biological activities of polysaccharides are continuing to be discovered. Some known activities include immune regulation, anti-tumor, lowering blood sugar and lipids, anti-virus, eliminating oxidative free radicals, and delaying aging. Polysaccharides are found in higher plants, animals, algae, and bacteria in nature and are widely distributed. Most of the polysaccharides are derived from tissue cells, which are less toxic and have fewer side effects on cells and the body, making them ideal drug sources. In recent years, with the rapid development of related disciplines such as biology and chemistry, polysaccharide compounds have also been increasingly studied. The international scientific community regards research on polysaccharides as the frontier of life sciences and has even proposed that the 21st century is the century of polysaccharides.

The Process of Polysaccharide Analysis

Extraction of polysaccharides

Polysaccharides are linked to the cell wall or interstitial substance by hydrogen or ionic bonds. Usually, different extraction methods are used based on whether or not polysaccharides are present and where the extraction is taking place. As solvents, hot water, acid, alkali, ethanol, etc. are often used, and microwaves or ultrasounds can help with the rough extraction process. In recent years, the most commonly used methods include supercritical fluid extraction technology and complex enzyme-assisted extraction technology. Using these methods, complex enzymes can specifically degrade the cell wall and the barrier for the dissolution of intracellular macromolecules in milder conditions, accelerating the release of polysaccharides. At the same time, the reaction can be controlled by changing the system conditions according to the characteristics of the enzyme.

Impurity removal from polysaccharides

Most crude polysaccharide extracts have impurities like inorganic salts, lipids, proteins, and low-molecular non-polar substances. For low-molecular-weight impurities, dialysis can be used to remove them. Protein is usually removed by the protease method, the Sevag method, the TCA method, or the trifluorothyl chloroethane method. Fat can be removed with organic solvents such as ethanol, ether, and petroleum ether. Adsorption and oxidation are common methods to remove pigment impurities.

Isolation and purification of polysaccharides

After separation and the removal of impurities, a mixed polysaccharide solution can be obtained, and the process to separate the mixed polysaccharide solution into various single polysaccharides is the purification of polysaccharides. More commonly used methods include sedimentation, chromatography, zone electrophoresis, ultracentrifugation, and other biochemical analysis methods. In general, polysaccharide purification requires the combination of two or more methods to optimize the results.

Analysis of polysaccharides

Polysaccharides have a wide variety of components, complex structures, and large molecular weights, so they are usually analyzed from the following four aspects.

1. Sugar content determination

In order to determine the sugar content in the sample, a developer-sulfuric acid method is often used. Monosaccharides, polysaccharides, and their derivatives are hydrolyzed to monosaccharides under the action of sulfuric acid and quickly dehydrated to form aldehyde derivatives, which are condensed with phenols, aromatic amines into colored compounds. The content of polysaccharides is indirectly determined by colorimetric quantification. These methods are simple, fast and sensitive, and have good color stability of colored compounds.

2. Molecular weight determination

There is no absolute method for determining the molecular weight of polysaccharides, so the method of statistical average is generally used. Osmotic pressure method, end group method, viscosity method, and light scattering method are commonly used in the past, but the operations are complicated and causing errors. Currently, more commonly used methods are gel filtration and high-performance liquid chromatography. These two methods must use standard polysaccharides of known molecular weight as a reference. For polysaccharides with a molecular weight of less than 50,000, mass spectrometry can be used.

3. Component determination

Polysaccharide composition analysis methods can be generally divided into: traditional chemical analysis, physical analysis (instrument analysis) and biological analysis. Among them, chemical analysis includes partial or complete acid hydrolysis, neutralization, and filtration. At the end, paper chromatography (PC), thin layer chromatography (TLC), gas chromatography (GC), liquid chromatography (HPLC), or ion chromatography are implemented for analysis. Many widely used instrumental analysis include spectrophotometry, infrared spectroscopy, nuclear magnetic resonance, GC, and mass spectrometry (MS).

4. Structure determination

Polysaccharides have more complex macromolecular structures than proteins. The variety of monosaccharides, connection methods, and the complexity of branch chains make the structure determination difficult. At present, the primary structure is the target for polysaccharide structure determination, analyzing mainly the molecular weight range of polysaccharides, the type, proportion, and connection order of monosaccharides, and configuration of glycosidic bonds. Common structural analysis methods are periodate oxidation, Smith degradation, and methylation reaction. Many advanced instruments used in recent years, such as ultraviolet, have greatly improved the analysis.

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