Polysacharides (PS), also known as polysaccharides, are composed of more than 10 monosaccharides linked by glycoside bonds（Oligosaccharides are formed by connecting less than 10 monosaccharides through glycoside bonds）, a natural macromolecular compound with a wide range of biological activities. It is widely distributed in higher plants, algae, microorganisms (bacteria and fungi) and animals. Since the 1960s, people have gradually discovered that polysaccharides have complex and multifaceted biological activities and functions:
(1) Polysaccharide can be used as a broad-spectrum immune enhancer, with immune regulation function, and can treat rheumatism, chronic viral hepatitis, cancer and other immune system diseases, and even anti AIDS virus. For example, Glycyrrhiza uralensis polysaccharide has obvious anti-virus and anti-tumor effects. Auricularia auricula polysaccharide, Ginkgo biloba exocarp polysaccharide and aloe vera polysaccharide can resist tumors and enhance human immune function.
(2) Polysaccharide has the functions of anti infection, anti radiation, anti coagulation, lowering blood sugar and blood lipid, and promoting the biosynthesis of nucleic acid and protein. For example, Bupleurum chinense polysaccharide has biological effects such as anti radiation and enhancing immune function, Ophiopogon japonicus polysaccharide has hypoglycemic and immune enhancing effects, and animal mucopolysaccharide has functions such as anticoagulation and lowering blood lipids.
(3) Polysaccharides can control cell division and differentiation, regulate cell growth and aging. For example, Parthenocissus polysaccharide has anti-virus and anti-aging effects, and Ginkgo biloba exocarp crude polysaccharide has anti-aging, anti allergy, blood lipid reduction, cough and expectorant, weight loss and other functions.
In addition, as a drug, polysaccharide has little toxicity, so the study of polysaccharide has aroused great interest.
Because the biological activity of polysaccharide is closely related to its structure, and the structure of polysaccharide is quite complex, the research in this field is relatively slow. However, people have done a lot of work in the separation, extraction and purification of polysaccharides.
1.Extraction method of polysaccharide
Bioactive polysaccharides mainly include fungal polysaccharides, plant polysaccharides and animal polysaccharides. The extraction of polysaccharides should first decide whether to do pretreatment before extraction according to the existing form and extraction position of polysaccharides. Animal polysaccharides and microbial polysaccharides are mostly surrounded by lipids. Generally, it is necessary to add acetone, ether, ethanol or a mixture of ethanol and ether for refluxing and degreasing to release polysaccharides. Attention should be paid to the roots, stems, leaves, flowers, fruits and seeds with high fat content when extracting plant polysaccharides. Before extraction, the raw materials should be degreased with low polarity organic solvents. At present, the extraction methods of polysaccharides mainly include solvent extraction, biological extraction, enhanced extraction, etc.
Chengdu biopurify pharmaceuticals Ltd. provides polysaccharides to customers,oligosaccharideThe extraction, separation, purification and identification services are welcome to consult.
1.1Solvent method
1.1.1 water extraction and alcohol precipitation method
Water extraction and alcohol precipitation is the most commonly used method for extracting polysaccharides. Polysaccharide is a polar macromolecular compound. Water, alcohol and other polar solvents should be selected for extraction. When using water as solvent to extract polysaccharide, it can be extracted by hot water immersion or cold water immersion percolation. Then, after concentrating the extract, add ethanol to the concentrate to make the final volume fraction reach about 70%. Using the property that the polysaccharide is insoluble in ethanol, the polysaccharide can be precipitated from the extract. After standing at room temperature for 5 hours, the mass fraction and yield of the polysaccharide are high. The factors that affect the extraction rate of polysaccharide include the amount of water, extraction temperature, solid-liquid ratio, extraction time and extraction times.
The water extraction and alcohol precipitation method for extracting polysaccharides does not need special equipment. The production process is low-cost and safe. It is suitable for industrial production. It is a desirable extraction method. However, due to the large polarity of water, it is easy to extract water-soluble components such as proteins and glycosides, which makes the extract corrupt during storage and brings difficulties to subsequent separation. In addition, this method is time-consuming and the extraction rate is not high.
1.1.2 acid extraction
In order to improve the extraction rate of polysaccharide, acid extraction method was developed on the basis of water extraction and alcohol precipitation method. For example, some polysaccharides containing acidic groups such as glucuronic acid are difficult to dissolve at low pH value. Acetic acid or hydrochloric acid can be used to make the extract acidic, and then ethanol can be added to precipitate the polysaccharides. Copper salt can also be added to form insoluble complexes or salt precipitates.
Because the existence of H + inhibits the dissolution of acidic impurities, the purity of polysaccharide products extracted by dilute acid extraction method is relatively high, but under acidic conditions, it may cause the fracture of glycosidic bond in polysaccharide, and the acid will cause corrosion to the container, so it is generally not suitable to use it except weak acid. Therefore, acid extraction also has some shortcomings.
1.1.3 alkali extraction
Polysaccharide is stable in alkaline solution. Alkali is conducive to the leaching of acidic polysaccharide, which can improve the yield of polysaccharide and shorten the extraction time. However, the extraction solution contains other impurities, which makes the viscosity too high and it is difficult to filter. Moreover, the extraction solution has a strong alkali taste and the color of the solution is yellow, which will affect the flavor and color of the finished product.
1.1.4 supercritical fluid extraction
Supercritical fluid extraction (SFE) is a new extraction and separation technology developed in recent years. Supercritical fluid refers to the state when substances are above the critical temperature and pressure. This fluid has the characteristics of both liquid and gas. It has high density, low viscosity, high solubility, penetrates into the matrix of extraction materials, and plays a very effective extraction function. Moreover, the solubility increases with the increase of pressure. After extraction, it is released by decompression. It has the advantages of maintaining the activity of active ingredients and no solvent residue. As the supercritical conditions of CO2 (TC = 304.6 ℃, TP = 7.38 MPa) are easy to reach, the solvent commonly used for supercritical extraction, supercritical CO2 at the pressure of 8 ~ 40 MPa is sufficient to dissolve any non-polar and medium polar compounds, and polar compounds can be dissolved after adding modifier.
The disadvantages of this method are complex equipment, high operation cost and limited extraction range.
1.2Enzymolysis
1.2.1 single enzyme hydrolysis
Single enzymatic hydrolysis refers to the biotechnology that uses an enzyme to extract polysaccharides, so as to improve the extraction rate. The commonly used enzymes include protease, cellulase, etc. Protease can decompose the free proteins in plant cells and make their structures loose; Protease can also hydrolyze the free protein in glycoprotein and proteoglycan, reduce their binding force to raw materials, and facilitate the extraction of polysaccharides.
1.2.2 complex enzymatic hydrolysis
A certain proportion of pectinase, cellulase and neutral protease are used for compound enzymatic hydrolysis. Cellulase and pectinase are mainly used to hydrolyze cellulose and pectin, breaking the cell wall of plant tissue cells and releasing the active polysaccharides in the cell wall. The amount of polysaccharides released is directly related to the amount of compound enzyme, enzymatic hydrolysis temperature, enzymatic hydrolysis time and enzymatic hydrolysis pH value.
The essence of enzymatic extraction is to strengthen the mass transfer process through enzymatic hydrolysis reaction. This method has the advantages of mild conditions, easy removal of impurities and high yield.
1.3Physical strengthening method
1.3.1 microwave assisted extraction
Microwave extraction is that high-frequency electromagnetic wave penetrates the extraction medium and reaches the interior of the extracted material, which can be quickly converted into heat energy to rapidly increase the internal temperature of the cell. The internal pressure of the cell exceeds the bearing capacity of the cell wall, the cell breaks, the effective components in the cell flow out, and dissolve in the extraction medium at a lower temperature. Through further filtration and separation, the extracted material is obtained.
Compared with other extraction methods, microwave-assisted extraction of polysaccharide has the advantages of high efficiency, simple operation, no introduction of impurities, high purity of polysaccharide, low energy consumption, low operating cost, and meets the requirements of environmental protection. It is a good polysaccharide extraction method.
1.3.2 ultrasonic assisted extraction
Ultrasonic extraction uses the mechanical effect, cavitation effect and thermal effect of ultrasonic. Mechanical effect can increase the movement speed and penetration of the medium, and can effectively break biological cells and tissues, so that the extracted effective components can be dissolved in the solvent; Cavitation effect makes the whole organism rupture, and the whole rupture process is completed in an instant, which is conducive to the dissolution of effective components; The thermal effect increases the dissolution rate of the active ingredients, which is instantaneous and can keep the biological activity of the extracted ingredients unchanged as much as possible; In addition, many secondary effects can also promote the dissolution of effective components in the extraction materials and improve the extraction rate.
Compared with the method of water boiling and alcohol precipitation, ultrasonic extraction is more effective and the extraction time is shorter; Compared with soaking method, the extraction rate was higher.
1.3.3 high voltage pulse method
The high-voltage pulse method is to repeatedly apply high-voltage short pulses (typically 20 ~ 80 kv/cm) to the flowing materials between the two electrodes for treatment. There are many hypotheses on the mechanism, such as cell membrane perforation effect, electromagnetic mechanism model, viscoelastic polarity formation model, electrolytic product effect, ozone effect, etc. the most studied is cell membrane perforation effect. The cells of animals, plants and microorganisms generate cross membrane potential under the action of external electric field. The insulating biofilm forms micropores due to the electric field, and the permeability changes. When the entire membrane potential reaches the limit value (about 1 V), the membrane breaks, the membrane structure becomes disordered, forming micropores, and the permeability increases. When the potential difference reaches the critical point, the cell breaks down.
2.Isolation and purification of Polysaccharides
2.1 Deproteinization
2.1.1sevage method
According to the denaturation characteristics of proteins in organic solvents such as chloroform, V (chloroform) ∶ V (amyl alcohol or n-butanol) was used as 5 ∶ 1 or 4 ∶ 1, the mixture was shaken violently for 20 ~ 30 min, the proteins were denatured into gels, centrifuged, and the denatured proteins at the junction of water layer and solvent layer were separated. This method can only remove a small amount of protein, which is inefficient and needs to be repeated for many times, resulting in loss of polysaccharide. However, this method is mild and has a good effect in avoiding polysaccharide degradation. If some protein hydrolases are added, the Sevage method is better. This method cannot remove lipoproteins because lipoproteins are soluble in chloroform.
2.1.2 trifluoro trichloroethane method
Mix the polysaccharide solution with trifluoro trichloroethane in the same volume, stir at low temperature for about 10 minutes, and centrifuge to obtain the upper water layer. The water layer continues to be treated repeatedly with the above method for several times to obtain a protein free polysaccharide solution. This method is more efficient than the seavg method, but the solvent has a low boiling point and is volatile, so it is not suitable for large-scale application.
2.1.3 trichloroacetic acid method
Trichloroacetic acid is a kind of organic acid, which denatures and precipitates the protein in the polysaccharide extract by interacting with the organic acid. This method is to add 5% ～ 10% trichloroacetic acid with the same volume as the polysaccharide aqueous extract to the polysaccharide aqueous extract, mix it evenly and let it stand overnight, centrifuge to remove the colloidal precipitation, and repeat the above operations until the solution is no longer turbid, so as to obtain the protein free polysaccharide. The higher the concentration of trichloroacetic acid, the better the effect of protein removal, but also the greater the impact on polysaccharides. It may be that trichloroacetic acid has a destructive effect on the structure of polysaccharides, causing the degradation of polysaccharides, and this destructive effect increases with the increase of trichloroacetic acid concentration.
Trichloroacetic acid method is often used to remove protein from plant polysaccharides, or protein hydrolase can be used first to partially degrade the protein in the sample, and then sevag method is used for better effect; Sevag method and trifluoro trichloroethane method are often used to remove proteins from microbial polysaccharides; It can also be removed by salting out and organic solvent extraction.
2.2Separation of Polysaccharides
It mainly includes fractional precipitation, quaternary ammonium salt precipitation, metal salt precipitation, chromatographic separation, membrane separation, dialysis, electrodialysis, etc. at present, DEAE gel or other different types of gel column chromatography and ion exchange chromatography are mostly used.
2.2.1 graded precipitation method
Most of the active polysaccharides are soluble in water, and the polysaccharides with less than 3 carbons are also soluble in ethanol. With the increase of polymerization degree, the polysaccharides
The solubility in ethanol decreases gradually. According to this property, ethanol can be added to the concentrated aqueous solution of polysaccharides in batches to gradually increase the volume concentration of ethanol to 50100200900 ml / L, so that polysaccharides with different degrees of polymerization can be precipitated respectively.
2.2.2 chromatographic separation
There are two commonly used chromatographic separation methods: gel column chromatography and ion exchange chromatography.
2.2.3 membrane separation method
Membrane separation technology (MST) is an efficient separation technology. In the separation process, the permeable membrane is selected as the separation medium. By applying a certain driving force (pressure difference, chemical potential difference, potential difference, etc.) on both sides of the membrane, the components in the raw liquid can selectively pass through the membrane. At present, ultrafiltration and microfiltration technology are widely used.
3Analysis and identification of Polysaccharides
3.1Determination of content
Determination methods: sulfuric acid phenol method, sulfuric acid anthrone method, colorimetric quantitative method, spectrophotometry, paper chromatography, ion exchange chromatography, yaphe method, thin layer chromatography, enzyme method, atomic absorption method, HPLC method, gel electrophoresis, affinity electrophoresis, continuous flow analysis [44], hypoiodate quantitative method, anthrone sulfuric acid method (total sugar), DNS method (reduction method), phosphorus molybdenum colorimetry, o-potassium aniline colorimetry, etc. Each method is only good for the measurement of some polysaccharides. Colorimetry, spectrophotometry, ion exchange chromatography, enzyme method and electrophoresis can be used for qualitative and quantitative analysis of polysaccharides at the same time.
3.2Purity identification
Polysaccharide is a high molecular compound, and its pure product is heterogeneous on the micro level. Generally speaking, pure polysaccharide is actually a homogeneous component with a certain molecular weight range. Common methods for purity identification of polysaccharides: ultracentrifugation, high pressure electrophoresis, gel chromatography, HLPC method, etc. HLPC method is widely used now, and the optical rotation measurement is also a method for purity measurement.
3.3Determination of molecular weight
The determination of molecular weight of polysaccharides is an important work to study the properties of polysaccharides. Common methods: osmotic pressure method, vapor pressure penetrant method, end group method, viscosity method, light scattering method, gel chromatography, excess rate method, precipitation method, gel electrophoresis method, HPLC method, ultracentrifugation analysis method, molecular sieve chromatography method, GPC method, MALDI-TOF-MS method.
3.4Structural determination
3.4.1 primary structure determination of polysaccharide
The primary structure analysis of polysaccharides is mainly to analyze the type, number, connection mode and glycoside bond configuration of monosaccharides. Chemical method and instrumental analysis method are commonly used. Determination of polysaccharide components and molecular proportion: partial acid hydrolysis, complete enzyme hydrolysis, chromatography; Analysis of the structure of pyran and furan rings: IR spectra; Connection sequence: selective spectroscopy, glycosidic bond sequence hydrolysis, nuclear magnetic resonance; α - β - epimer: glycosidase hydrolysis, NMR; Substitution of hydroxyl groups: methylation reaction, gas chromatography, periodate oxidation, Smith degradation method, Terho method, nuclear magnetic resonance, mass spectrometry; The connection mode of sugar chain and peptide chain: monosaccharide and amino acid composition, dilute alkali hydrolysis, hydrazinolysis reaction; There are many methods to analyze the structure of polysaccharides. So far, there is no single method to analyze the structure of polysaccharides. The combination of instrumental analysis and chemical methods is a common method for the determination of polysaccharide structure.
3.4.2 determination of higher structure of Polysaccharides
At present, the common means to study the secondary structure of polysaccharides is NMR technology, such as 2D-NMR, 13C spectrum. The general method is to combine modern NMR technology with theoretical calculation to screen the conformation through certain theoretical calculation. The main theoretical calculation methods include ab initio calculation, abundance empirical calculation and empirical force field calculation. Circular dichroism (CD) can also be used to analyze the conformation of sugars. In recent years, it has reached an unprecedented depth and breadth to reveal the laws of important life activities based on accurate three-dimensional structure knowledge. As an important class of bioactive macromolecules, the study on the structure of polysaccharides is bound to promote the further development of the understanding of polysaccharides.
Chengdu biopurify pharmaceuticals Ltd. provides polysaccharides to customers,oligosaccharideThe extraction, separation, purification and identification services are welcome to consult.