Polysaccharides are one of the four basic substances that constitute life, and widely exist inhigher plant、animal、microorganism、LichensandseaweedSuch as plant seeds, stem and leaf tissues, animal mucus, insect and crustacean fungi, bacteria, etc.Polysaccharides play an important role in anti-tumor, anti-inflammatory, anti-virus, hypoglycemic, anti-aging, anticoagulant, immune promotion, etcbiological activityeffect.Polysaccharide with immunological activityderivativeThere are often otheractivityFor example, sulfated polysaccharides have anti HIV activity and anticoagulant activity, and carboxymethyl polysaccharides have anti-tumor activity.Therefore, the research and development of polysaccharides has attracted more and more attention.
Chinese name
Polysaccharides
Foreign name
polysaccharide
Nature
One of the four basic substances constituting life
At present, the declaration of new polysaccharide drugs is increasing.Therefore, it is necessary to explore the pharmaceutical research and evaluation of new polysaccharide drugs.The structure of polysaccharide drugs is extremely complex, and the pharmaceutical research and evaluation is still difficult. This paper, based on the existing research level, conducts a preliminary exploration of its pharmaceutical evaluation.
Extraction method
Announce
edit
Extraction and purification of polysaccharides or microbial intracellular polysaccharides existing in animals and plants, because most of them havelipidThe first step to release polysaccharide is to remove the surface lipid, and alcohol or ether is usually used for refluxDegrease。
Extract the degreased residue with water based solution to take polysaccharide (i.e. cold water, hot water, hot or cold 0.1-1.0mol/L NaOH, hot or cold 1%acetic acidOr 1%phenolThe polysaccharide extract thus obtained contains many impurities, mainlyinorganic salt, Lowmolecular weightOfOrganic matterAnd high molecular weightprotein、LigninEtc.
To remove these impurities, it can be used for inorganic salts and low molecular weight organic substancesDialysis、ion exchange resinorGel filtration methodexcept;Available for macromolecular impuritiesenzymeDigestion (such as protease and lignin enzyme), solvent precipitation method such as ethanol or acetone or metal complex method.Removal of protein from polysaccharide extract is a very important step. The commonly used methods are Sevag method, trifluorotrichloroethane methodTrichloroacetic acidThe latter is more intense, and should not be used for polysaccharides containing furan residues due to unstable connecting bonds.However, this method is efficient and easy to operate. It is often used for polysaccharide from plants.None of the above three methods is suitable forGlycopeptideBecause glycopeptides will also precipitate like proteins.After protein removal, dialysis should be performed again, and different specifications should be selectedultrafiltration membraneandDialysis bagconductUltrafiltrationAnd dialysis, which can separate and purify polysaccharides with different molecular sizesSmall moleculeThe material is very practical and can meet the needs of mass production.It has broad application prospects.
So far, the obtained extract is basically a polysaccharide mixture without protein and small molecular impurities.Generally speaking, the mixture obtained by the above method is a polysaccharide mixture. If a single polysaccharide is to be obtained, the mixture must also be purified.
column chromatographyIt is commonly used in the purification of polysaccharides and is often divided into two categories: one is gel column chromatography with molecular sieve only, which can achieve the separation purpose according to the size and shape of polysaccharide molecules. The commonly used gels areDextran gelandagarose gel, and Sephacryl with better performance.EluentIt refers to salt solution and buffer solution of various concentrations, and its ionic strength shall not be less than 0.02mol/L.SecondIon exchange chromatographyIt not only has the function of molecular sieve according to the different molecular weight, but also has the function of molecular sieve. The common exchange agents are DEAE cellulose, DEAE glucan and DEAE-agaroseThis method is suitable for separating various acidic, neutral polysaccharides and mucopolysaccharides.
Other methods can also be used for the purification of polysaccharides, such as preparativeHigh performance liquid chromatographyPreparationZone electrophoresis, affinity chromatography, etc. These methods are sometimes very useful for preparing some small amounts of pure products for analysis.
Quality research
Announce
edit
Generally speaking, the quality research of polysaccharides mainly includes the physical and chemical properties of each component, such as solubility, specific rotation andviscosityDetermination, molecular weight andmolecular weight distributionResearch on plane and three-dimensionalChemical structureAnalysis, research on structural modification and structural modification, andUronic acid, proteinmonosaccharideAnd polysaccharide content determination, etc.The structure, molecular weight, molecular weight distribution and content determination of polysaccharides are briefly introduced below.
At present, chemical and physical methods are mostly used in the analysis of the primary structure of polysaccharides, which can basically clarify the general characteristics of the primary structure of a polysaccharide.At present, physical methods, such as X-ray fiber diffraction, nuclear magnetic resonance, and electron diffraction, are mainly used to analyze the advanced structure of polysaccharides.As mentioned above, the primary structure of polysaccharide itself is very complex.Due to the microscopic heterogeneity of polysaccharide structure, or defects in structural bonds, or molecular weight dispersion, it is difficult to obtain completely correct structural formula from the primary structural analysis of polysaccharides.The description of polysaccharide structure includes: ① molecular weight range of polysaccharide; ②Monosaccharide component of polysaccharide; ③Type of monosaccharide junction; ④Configuration of monosaccharide and glycosidic bond; ⑤Duplicate units.The activity of polysaccharides is closely related to their primary and advanced structures, and the advanced structures play a greater role in activity than the primary structures.Some polysaccharides have the same primary structure but different activities. The reason is that the secondary and tertiary structures are different.At present, the three-dimensional structure of polysaccharides is generally studied by 2D-NMR and X-ray diffraction.In addition, the activity of polysaccharide is also related to its molecular weight, solubility, viscosity and other physical and chemical properties.When studying the structure-activity relationship of polysaccharides, we often use molecular modification of polysaccharides and chemical modification of polysaccharides, such as sulfation, desulfurization acidification, chemical degradation, enzymatic degradation, acetylation, alkylation, etc., which is helpful to further explore their structure-activity relationship.
Analytical method
Announce
edit
Chemical methods and physical analysis methods will be briefly introduced below. ⑴Chemical method is still the most commonly used method to determine the structure of polysaccharides. There are many methods for determination, among which the classic and effective ones are methylation analysis, periodate oxidation and Smith degradation, partial acid hydrolysis, acetylation and methanolysis. ①
Methylation analysis
Methylation analysis is one of the most powerful means for structural analysis of polysaccharides and oligosaccharides.It includes that all free hydroxyl groups of sugars generate methyl ether, then methylated monosaccharides are released through hydrolysis, and then reduced to sugar alcohols through NaBH4, and then acetylated the hydroxyl groups generated after hydrolysis to obtain various partially methylated sugar alcohol acetyl derivatives. The generated products are qualitatively and quantitatively analyzed by gas chromatography to determine the types and proportions of monosaccharides that make up polysaccharides,Furthermore, the attribution of various partially methylated monosaccharide derivatives can be obtained by gas chromatography-mass spectrometry combined with the analysis of standard spectrogram, so as to determine the connection position of each monosaccharide, that is, the position of glycosidic bond.However, the methylation analysis is still unable to know the isomeric glycoside bond configuration and the sequence information of monosaccharide residues in polysaccharides. What should be noted is that it is difficult to methylate polysaccharides containing uronic acid or aminohexose residues, and it may produce secondary products, such as uronic acid residues can produce ketal derivatives,N-methyl-N-acetylaminohexose can be produced by N-acetylaminohexose residues, and these derivatives need special analytical techniques to identify.
Periodate oxidation
The periodate oxidation reaction of polysaccharides is usually carried out in the aqueous solution of pH3-5, using periodate as oxidant. Because the oxidation products of dialdehyde type are unstable in water, they need to be reduced to alcohol with NaBH4 before acid hydrolysis. Finally, the analysis results of hydrolysis products show that the types of monosaccharide connections in polysaccharides are l → 4, 1 → 6, 1 → 2, or all kinds of connections.
Smith degradation
Smith degradation is actually a kind of improved periodate oxidation, which is to oxidize polysaccharide periodate, reduce NaBH4, and then partially hydrolyze it with weak acid (usually hydrolyze the reduction product with dilute inorganic acid at room temperature) to generate characteristic sugar linked repeating units, so as to obtain more structural information. ③Partial acid hydrolysis is a very useful technique in the structural analysis of polysaccharides. First, short chain segments whose structure is easier to determine can be obtained through partial acid hydrolysis, so that the structure of polysaccharides can be inferred from zero.Second, it can break at the special glycoside bond to help the whole structure analysis.If the monosaccharide with furan ring structure is unstable to acid, it can be hydrolyzed with weak acid (these residues can be obtained, and then useful structural data can be obtained through the analysis of residue fragments).A successful example is a medlar glycoproteinArabinoseO-linked polysaccharide composed of and galactose. From the structure of methylation analysis, it is known that the non reducing end of the polysaccharide is furan cyclic arabinose. Through partial acid hydrolysis and paper chromatography tracking detection, Ara is released first, and hydrolysis is terminated when Ga1 is just released. Through comparison of methylation analysis results of the two polysaccharides before and after hydrolysis,Combined with the results measured by other methods, the possible structure of the whole polysaccharide can be inferred.
Acetolysis and methanolysis
Acetolysis: The acetylation reaction of polysaccharides is carried out by heating in a mixed solution composed of acetic anhydride, acetic acid and sulfuric acid, and is cracked at a certain glycosidic bond.The results show that the speed of acid hydrolysis and acetylation of the same glycoside bond is different.Acetolysis is a useful supplement to acid hydrolysis. Polysaccharides can obtain different fragments from these two different methods and obtain structural information of polysaccharides from different angles.
Methanolysis: the reaction of polysaccharide with anhydrous methanol hydrogen chloride at 80-100 ℃ can change the polysaccharide into methyl glycosides that make up monosaccharides. These methyl glycosides can be converted into trimethylsilyl ether derivatives or acetyl derivatives, and then GC analysis and comparison with standard monosaccharides can obtain quantitative data of monosaccharides that make up polysaccharide. ⑵Physical analysis method ① IR method: IR is mainly used to determine the glycosidic bond configuration of pyranose and routinely observe other functional groups in polysaccharide structure analysis.Generally, the scope of 730-960cm-1 is mainly observed. For α - pyranose, δ C1-H is 845 cm-1, while for β - pyranose, δ C1-H has the maximum absorption peak at 890 cm-1. ②MS, GC-MS: GC analysis of polysaccharides is limited by the volatility and thermal stability of the sample, but GC-MS is an indispensable tool for the structural analysis of polysaccharides, especially for the analysis of hydrolyzed monosaccharides, methylated monosaccharides and methylated oligosaccharides, and can identify the isomers of sugars.MS is not only essential to identify the fragments of various methyl derivatives and determine the connecting positions of various monosaccharide residues in the structural analysis of polysaccharides, but also can be used to determine the molecular weight and primary structure of polysaccharides due to the emergence of FAB-MS, ESI-MS and MALDI-MS. ③NMR: One of the characteristics of using NMR technology to study the structure of polysaccharide is that it does not destroy the sample. The structural characteristics of polysaccharide can be expressed by chemical shift, coupling constant, integral area, NOE, relaxation time and other parameters.One and two dimensional NMR spectra have broad application prospects in the analysis of sugar configuration, position and order of interconnects.
2. Molecular weight and molecular weight distribution Polysaccharides have the characteristics of uneven molecular size. In recent years, it has been found that one of the molecular weight components of these biomacromolecules has pharmacological activity, while the other has no pharmacological activity or has certain toxic side effects,Therefore, molecular weight and its distribution are both indicators of effectiveness control and indicators of safety control of such drugs. It is very necessary to develop this inspection in quality standards, which is also an obvious feature of the development of quality standards for macromolecular polymer drugs in recent years.The molecular weight of polysaccharide only represents the average distribution of similar chain length. The molecular weight measured by different methods is different, even for the same polysaccharideWeight average molecular weightThere is also a large difference with the number average molecular weight, usually usingGel chromatographyTo control the molecular weight and distribution of such drugs, the chromatographic column filler suitable for the molecular size of the test article should be selected through research;The mobile phase used is usually water or buffer solution. Its pH value should not exceed the tolerance range of the filler. An appropriate amount of organic solvent can be added, but the concentration should not exceed 30%. The flow rate should be 0.5-1.0ml/min. Since most of these molecules have no UV absorption, they are generally usedDifferential refractometerThe molecular weight range and particle shape of the selected reference substance should match the test substance, and the measured data should be processed by appropriate GPC software to obtain relevant parameters.
3. Generally speaking, the polysaccharide does not contain protein andamino acidThe protein or amino acid test should be negative or meet the limit inspection requirements. If it is glycoprotein or glycopeptide, the evidence should be provided to ensure that the product is not a mixture of polysaccharide and protein;It also provides its amino acid composition and protein content range to ensure stable and controllable quality.It is of great significance to determine whether there are uronic acid residues in polysaccharide obtained from natural plants, especially in the structural analysis of sugar composition.At present, carbazole sulfate method is commonly used to determine the content of uronic acid, but it is easily interfered by neutral sugar residues.In order to eliminate the interference of determination, the absorbance of neutral sugar in the sample can be measured first, and then the absorbance of neutral sugar can be subtracted from the absorbance of the sample, which is the absorbance value of uronic acid in the sample.M-hydroxybiphenylThis method is also a commonly used method for the determination of uronic acid in polysaccharides, which is less interfered by neutral sugar residues than carbazole sulfate method.
The content determination of polysaccharide can be divided into two categories: one is to directly determine the polysaccharide itself, such as HPLC and enzyme method;The other is the method established by the condensation reaction of monosaccharides that make up polysaccharides, such asPhenol sulfuric acid method, anthrone sulfuric acid method, etc.The former requires pure polysaccharides and specific enzymes, while the latter has great methodological interference in determination, poor colorimetric reproducibility and many influencing factors.However, due to the current domestic experimental conditions, this method is still mainly used for the content of polysaccharides. Its principle is: polysaccharides are rapidly hydrolyzed under the high temperature produced by the hydration of concentrated sulfuric acid to produce monosaccharides, and monosaccharides react with phenol under strong acid conditions to produce orange derivatives.At the wavelength of about 490nm and within a certain concentration range, the absorption value of the derivative has a linear relationship with the concentration of monosaccharide, so its content can be determined by colorimetry. The monosaccharide reference substance used should be monosaccharide with the same composition or higher content as its polysaccharide, so the measured value is more accurate.It should be emphasized that this method determines the total sugar content rather than the total polysaccharide content, so first determine the free monosaccharide content in the sample, and then subtract the free monosaccharide content from the total sugar content, which is the total polysaccharide content.In addition, the 3,5-dinitrosalicylic acid colorimetry (DNS method) can also be used. It is developed under alkaline conditions. The content of reducing sugar and total sugar can be measured accurately to calculate the content of polysaccharide, which can eliminate the interference of reducing impurities.
epilogue
Announce
edit
In the past 20 years, due to the rapid development of biology, chemistry and other disciplines, people have more and more in-depth understanding of the active role of polysaccharides and their compounds, but there are still many problems to be further studied and solved in polysaccharide itself. For example, some polysaccharide substances have very low content in natural products, which is difficult to extract and separate, and some polysaccharide quality is not easy to control, especiallyFungus plantPolysaccharide extracted and separated after fermentation.The determination of polysaccharide structure has its own difficulty and particularity, and the current research level and level are still relatively backward.Therefore, it is necessary for the applicant to conduct a lot of basic research in the research and development of new polysaccharide drugs.
