CN101441202A - Method for detecting fungal polysaccharide - Google Patents
Method for detecting fungal polysaccharide Download PDFInfo
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- CN101441202A CN101441202A CNA2008102315392A CN200810231539A CN101441202A CN 101441202 A CN101441202 A CN 101441202A CN A2008102315392 A CNA2008102315392 A CN A2008102315392A CN 200810231539 A CN200810231539 A CN 200810231539A CN 101441202 A CN101441202 A CN 101441202A
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Abstract
The invention discloses a method for detecting fungi polysaccharide. The method organically integrates MALS, high-response differential method, and DLS to carry out online, offline, and quantitative detection, with monophase chromatographic technique as core, thus, the characteristics of polysaccharide, like absolute molecular weight, dispersion degree, molecular conformation, dissolubility, and stability, can be represented and analyzed. The method comprises preparation of sample, preparation of mobile phase, sample injection, buffering, focusing balancing, elution and separation, detection, and the like. Compared with prior phenol-sulfuric acid method, gel electrophoresis method, and HPLC method, the invention has the advantages that quantitative detection and qualitative detection are integrated; the sample is not damaged through whole physical detection; the sample adsorption quantity is little; the operation is simple and rapid, and the condition is mild; the absolute molecular weight and the distribution thereof of the polysaccharide can be obtained without standard sample; the sensitivity is high and can reach 10<-9>; and the physicochemical characteristics of polysaccharide can be simultaneously detected. The invention can be widely applicable to analysis detection, quality control, research and development of polysaccharide and other related compounds.
Description
Technical field
The present invention relates to a kind of method that detects fungi polysaccharide.
Background technology
Polysaccharide is the abundantest macromolecular compound of nature content, and it is extremely extensive in distributed in nature, and existence is all arranged in higher plant, algae, mushroom and the animal body, and various biological that polysaccharose substance had and pharmacological function have caused people's great attention.The subject that the polysaccharide science was a dark horse as the genome times afterwards comprehensively has been included international forward position research field in.In the world, food, medicinal fungi glycocalix are called " biological response modifier " (Biological Response Modifier, be called for short BRM), because it is the bioactivator that a class can strengthen immune function of human body, have physiology and pharmacological function widely, in recent years, food, medicinal fungi polysaccharide have become one of the research focus in fields such as biological chemistry, molecular biology, medical science, pharmacy, Food Science.Though people have carried out comparatively extensive research to aspects such as the structure of fungi polysaccharide, biologically active, pharmacological functions, how the efficient detection fungi polysaccharide is not also seen suitable method report.At present, employed polysaccharide detection method mainly contains phenolsulfuric acid method, gel electrophoresis, HPLC method etc.The phenolsulfuric acid method is a kind of classical way that detects polysaccharide, and being referred to as again is the standard method of detection by quantitative polysaccharide, but this method be a kind of can't strict quantitative chemical analysis, not only operative technique is difficult to grasp, and error is bigger.Gel electrophoresis and HPLC method can be described as the modernism of analyzing and testing polysaccharide, the use prerequisite of gel electrophoresis is that sample is charged, and most polysaccharide all are uncharged neutral compounds, and this just makes the usable range of gel electrophoresis be subjected to great restriction.The HPLC method is for a kind of really good method of most macromolecular compound analyzing and testing, but because the molecular weight of polysaccharide is bigger, and viscosity is big, the absorption of polysaccharide and stationary phase is a problem that is difficult to overcome all the time.In addition, these above-mentioned methods all also must need the reference material work with reference to using.
Summary of the invention
It is easy, rapid that technical matters to be solved by this invention provides a kind of running program, and mild condition is not destroyed sample, and sample absorption is few, need not standard specimen, and is highly sensitive, can detect the detection method of the multiple physicochemical characteristics parameter of fungi polysaccharide simultaneously.
The technical solution used in the present invention is:
The method of this detection fungi polysaccharide be with have broad dynamic range, do not have shearing force, the single-phase chromatographic technique that separates fast is core, and with the multi-angle laser light scattering method, but highly sensitive differential method and dynamic light scattering method carry out that organic assembling constitutes a kind of not only can be online, but also off-line, both but direct quantitative detected fungi polysaccharide, again can be to the absolute molecular weight of polysaccharide; Molecular dimension and second virial coefficient; Dispersion degree; The root mean square radius of turn; Molecular conformation; The method that characteristic such as dissolubility and stability characterizes and analyzes.
The method that this detects fungi polysaccharide specifically comprises following processing step:
(1) sample preparation
The fungi polysaccharide sample that takes by weighing 10-100mg places clean test tube or little measuring cup, adds 5-60ml distilled water or 0.85% sodium chloride solution or phosphate buffer (7.0mmol/L KH
2PO
4, 7.0mmol/LNa
2HPO
4, after pH=6.8) stirring makes its dissolving, in 4 ℃ of low temperature refrigerators, preserve standby.The concentration of sample liquid is 0.1-0.98mg/ml;
(2) preparation of moving phase
The above-mentioned solvent that is used to dissolve polysaccharide outgased with degasser handle the back as moving phase;
(3) sample introduction, buffering, focusing balance
With single-phase chromatogram (asymmetric field flow point from) instrument (The Eclipse AF4), multi-angle static light scattering instrument (MALS, DAWN HELEOS II), dynamic light scattering (DLS, DynaPro Titan), differential refraction detector (RI, OPTILAB rEX), degasser (1100 Series vacuum degasser) and sampling pump etc. effectively are connected and are integrated into a complete system; With this system is the method that this detection fungi polysaccharide is implemented on the basis;
Process condition: carry out sample introduction, buffering and focus on balance with micropipettor above-mentioned polysaccharide sample 5-120 μ L that has prepared of absorption and an amount of moving phase.Sample introduction, buffering, focusing equilibration time are 5-10min, sample introduction speed: 0.05-0.95mL/min, and the focusing equilibration time is 1-5min; Runner flow velocity: 0.2-2mL/min, the cross-current flow velocity: initial with 2-9mL/min operation 1-5 minute, to reduce to the 1-4mL/min operation then and be reduced to 0ml/min gradually after 3-8 minute, total run time is 10-60 minute; Mode of operation is to regulate the flow velocity of the moving phase of runner stream and cross-current earlier, begins sample introduction again after ready to balance is stable.The flow velocity of sugar sample is: 0.05-0.95mL/min;
Sample introduction had both comprised that the auto injection mode also comprised the hand sampling mode, buffering and focus on balance be both comprised runner stream and cross-current moving phase, also comprise the buffering and the focusing balance of polysaccharide sample.
(4) wash-out separates
Moving phase solution with above-mentioned preparation is made eluant, eluent, begins wash-out 5-49 minute after above-mentioned sample feeding, buffering and focusing balance are finished;
(5) detect
Detection means adopts online or two kinds of detections of off-line, and online detection is meant from wash-out and begins to detect to the whole process that wash-out finishes; Offline inspection then is meant the whole detections after wash-out finishes.Detect the not only water-soluble but also water-insoluble polysaccharide of the kind of polysaccharide.
Fungi polysaccharide sample of the present invention means any in food, the medicinal fungi polysaccharide such as lentinan, Hericium erinaceus polysaccharide, ganoderan, Blackfungus polyhexose, grifolan or several.Its method for making sample is a kind of method that does not change the sample physicochemical property.
The beneficial effect of detection fungi polysaccharide method disclosed in this invention is:
This method is the single-phase chromatographic technique to have broad dynamic range, not have shearing force, separate fast, and with the multi-angle laser light scattering method, but dynamic light scattering method and highly sensitive differential method are carried out a kind of both direct quantitative detection polysaccharide that organic assembling constitutes, again can be to the absolute molecular weight of polysaccharide; Molecular dimension and second virial coefficient; Dispersion degree; The root mean square radius of turn; Molecular conformation; The method that characteristic such as dissolubility and stability characterizes and analyzes.Compare with methods such as existing polysaccharide detection method such as phenolsulfuric acid method, gel electrophoresis, HPLC, the present invention has following characteristics: but 1. not only can be quantitatively but also the qualitative detection polysaccharide, and it is a kind of the collection quantitatively and qualitative detection method.2. do not destroy sample, full physical detection.3. sample absorption is few.4. running program is easy, rapid, mild condition.5. need not absolute molecular weight and distribution thereof that standard specimen can obtain polysaccharide, and the molecular weight magnitude range of the polysaccharide of surveying is extensive.6. highly sensitive, can reach 10
-97. can detect the multiple physicochemical characteristics of polysaccharide simultaneously.The present invention can be widely used in analyzing and testing, quality control and the research and development of polysaccharide and other related compound.
Embodiment
Below introduce embodiments of the invention.
Embodiment 1: the method that this detects fungi polysaccharide specifically comprises following processing step:
(1) sample preparation
Take by weighing polysaccharide sample (lentinan, Hericium erinaceus polysaccharide, the grifolan of a certain amount of (20mg) respectively, flammulina velutipes, bitter melon polysaccharide) place 5 clean test tubes or little measuring cup, adding 5ml distilled water is preserved standby in low temperature (4 ℃) refrigerator after stirring and making its dissolving.The concentration of sample liquid is 0.1mg/ml;
(2) preparation of moving phase
The above-mentioned solvent that is used to dissolve polysaccharide outgased with degasser handle the back as moving phase;
(3) sample introduction, buffering, focusing balance
With single-phase chromatogram (asymmetric field flow point from) instrument (The Eclipse AF4), multi-angle static light scattering instrument (MALS, DAWN HELEOS II), dynamic light scattering (DLS, DynaPro Titan), differential refraction detector (OPTILAB rEX), degasser (1100 Series vacuum degasser) and sampling pump etc. effectively are connected and are integrated into a complete system; With this system is that present embodiment and following each embodiment are implemented in the basis;
Process condition: carry out sample introduction, buffering and focus on balance with the micropipettor above-mentioned polysaccharide sample that has prepared 10 μ L of absorption and an amount of moving phase.Sample introduction, buffering, focusing equilibration time are 5min, sample introduction speed: 0.1mL/min; The focusing equilibration time is 1min, runner flow velocity: 0.2mL/min, and the cross-current flow velocity: initial with 2mL/min operation 5 minutes, to reduce to the 1mL/min operation then and be reduced to 0ml/min gradually after 8 minutes, total run time is 55 minutes.The flow velocity of sugar sample is: 0.1mL/min;
(4) wash-out separates
Moving phase solution with above-mentioned preparation is made eluant, eluent, begins wash-out 48 minutes after above-mentioned sample feeding, buffering and focusing balance are finished;
(5) detect
Detection means is online detection.
Embodiment 2: identical with the step of embodiment 1, different is:
1. take by weighing polysaccharide sample (lentinan, Hericium erinaceus polysaccharide, the grifolan of 60mg, flammulina velutipes, bitter melon polysaccharide) place clean test tube or little measuring cup, adding 30ml0.85% sodium chloride solution is preserved standby in low temperature (4 ℃) refrigerator after stirring and making its dissolving.The concentration of sample liquid is 0.5mg/ml;
2. the operating procedure condition is: carry out sample introduction, buffering and focus on balance with the micropipettor above-mentioned polysaccharide sample that has prepared 60 μ L of absorption and an amount of moving phase.Sample introduction, buffering, focusing equilibration time are 8min, sample introduction speed: 0.5mL/min; The focusing equilibration time is 3min, runner flow velocity: 1.2mL/min, and the cross-current flow velocity: initial with 5mL/min operation 3 minutes, to reduce to the 2.5mL/min operation then and be reduced to 0ml/min gradually after 5.5 minutes, total run time is 35 minutes.The flow velocity of sugar sample is: 0.5mL/min;
3. wash-out separates: the moving phase solution (0.85% sodium chloride) with above-mentioned preparation is made eluant, eluent, begins wash-out 23 minutes after above-mentioned sample feeding, buffering and focusing balance are finished;
4. the detection means of present embodiment is an offline inspection.
Embodiment 3: identical with the step of embodiment 1, different is:
1. the polysaccharide sample that takes by weighing 100mg places clean test tube or little measuring cup, adds 60ml phosphate buffer (7.0mmol/L KH
2PO
4, 7.0mmol/L Na
2HPO
4, after pH=6.8) stirring makes its dissolving, in low temperature (4 ℃) refrigerator, preserve standby.The concentration of sample liquid is 0.95mg/ml;
2. the operating procedure condition is: carry out sample introduction, buffering and focus on balance with the micropipettor above-mentioned polysaccharide sample that has prepared 120 μ L of absorption and an amount of moving phase.Sample introduction, buffering, focusing equilibration time are 10min, sample introduction speed: 0.95mL/min; The focusing equilibration time is 5min, runner flow velocity: 2.0mL/min, and the cross-current flow velocity: initial with 9mL/min operation 1 minute, to reduce to the 4mL/min operation then and be reduced to 0ml/min gradually after 3 minutes, total run time is 25 minutes.The flow velocity of sugar sample is: 0.95mL/min;
3. wash-out separates: the moving phase solution (phosphate buffer) with above-mentioned preparation is made eluant, eluent, begins wash-out 15 minutes after above-mentioned sample feeding, buffering and focusing balance are finished;
4. the detection means of present embodiment is online detection.
According to above-mentioned 3 embodiment, all can obtain following result (see Table 1, table 2):
Table 1: the concentration of several not homopolysaccharide samples and molecular weight (Mw)
Table 2: the concentration of several not homopolysaccharide samples and molecular weight distribution characteristic (Mw)
Claims (7)
1. method that detects fungi polysaccharide comprises following processing step:
(1) sample preparation
The fungi polysaccharide sample that takes by weighing 10-100mg places clean test tube or little measuring cup, adds 5-60ml distilled water or 0.85% sodium chloride solution or phosphate buffer (7.0mmol/L KH
2PO
4, 7.0mmol/LNa
2HPO
4, pH=6.8) stir make its dissolving after, in 4 ℃ of low temperature refrigerators, preserve standbyly, the concentration of sample liquid is 0.1-0.98mg/ml;
(2) preparation of moving phase
The above-mentioned solvent that is used to dissolve polysaccharide outgased with degasser handle the back as moving phase;
(3) sample introduction, buffering, focusing balance
With single-phase chromatogram (asymmetric field flow point from) instrument (The Eclipse AF4), multi-angle static light scattering instrument (MALS, DAWN HELEOS II), dynamic light scattering (DLS, DynaPro Titan), differential refraction detector (RI, OPTILAB rEX), degasser (1100Series vacuum degasser) effectively is connected with sampling pump etc. and is integrated into a complete system, is the method that this detection fungi polysaccharide is implemented on the basis with this system;
Process condition: carry out sample introduction, buffering and focus on balance with micropipettor above-mentioned polysaccharide sample 5-120 μ L that has prepared of absorption and an amount of moving phase, sample introduction, buffering, focusing equilibration time are 5-10min, sample introduction speed: 0.05-0.95mL/min, the focusing equilibration time is 1-5min; Runner flow velocity: 0.2-2mL/min, the cross-current flow velocity: initial with 2-9mL/min operation 1-5 minute, to reduce to the 1-4mL/min operation then and be reduced to 0ml/min gradually after 3-8 minute, total run time is 10-60 minute; Mode of operation is to regulate the flow velocity of the moving phase of runner stream and cross-current earlier, begins sample introduction again after ready to balance is stable, and the flow velocity of sugared sample is: 0.05-0.95mL/min;
(4) wash-out separates
Moving phase solution with above-mentioned preparation is made eluant, eluent, begins wash-out 5-49 minute after above-mentioned sample feeding, buffering and focusing balance are finished;
(5) detect
Detection means adopts online or two kinds of detections of off-line, and online detection is meant from wash-out and begins to detect to the whole process that wash-out finishes; Offline inspection then is meant the whole detections after wash-out finishes.
2. the method for detection fungi polysaccharide according to claim 1 is characterized in that: the fungi polysaccharide sample in described (1) sample preparation means any in food, the medicinal fungi polysaccharide such as lentinan, Hericium erinaceus polysaccharide, ganoderan, Blackfungus polyhexose, grifolan or several.
3. the method for detection fungi polysaccharide according to claim 1 and 2 is characterized in that: described step (1) method for making sample is a kind of method that does not change the sample physicochemical property.
4. the method for detection fungi polysaccharide according to claim 1 and 2 is characterized in that: the moving phase in described (2) step is meant that all can dissolve the solvent of polysaccharide, comprises water and organic phase.
5. the method for detection fungi polysaccharide according to claim 1 and 2, it is characterized in that: the sample introduction in described (3) step had both comprised that the auto injection mode also comprised the hand sampling mode, buffering and focus on balance be both comprised runner stream and cross-current moving phase buffering and focus on balance, also comprise the buffering and the focusing balance of polysaccharide sample.
6. the method for detection fungi polysaccharide according to claim 1 and 2 is characterized in that: the kind that described (5) step detects polysaccharide not only can be a water-soluble polysaccharide but also can be water-insoluble polysaccharide.
7. the method for detection fungi polysaccharide according to claim 1 and 2, it is characterized in that: described (3), (4) and (5) step are with the piece-rate system of single-phase chromatogram as sample, and with multi-angle laser light scattering method (MALS), dynamic light scattering (DLS) method and highly sensitive differential (OPTILAB rEX) method is carried out the various physical and chemical parameters that dynamically online or offline inspection obtains polysaccharide; It is with single-phase chromatography, multi-angle laser light scattering method (MALS), dynamic light scattering (DLS) method and highly sensitive differential (OPTILAB rEX) but method carry out a kind of both direct quantitative that organic assembling constitutes and detect polysaccharide, again can be to the absolute molecular weight of polysaccharide; Molecular dimension and second virial coefficient; Dispersion degree; The root mean square radius of turn; Molecular conformation; The method that characteristic such as dissolubility and stability characterizes and analyzes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936901A (en) * | 2009-06-30 | 2011-01-05 | 怀亚特技术公司 | The characterizing method of macromolecular reversible association in the solution of high concentration |
| CN114605566A (en) * | 2022-03-14 | 2022-06-10 | 中国科学院长春应用化学研究所 | Method for extracting aloe gel macromolecule active ingredient and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1245623C (en) * | 2003-03-04 | 2006-03-15 | 南京康海药业有限公司 | Lentinan molecular weight and molecular weight distribution measuring method |
| CN1220876C (en) * | 2003-08-01 | 2005-09-28 | 莱阳农学院 | Method for detecting active polysaccharide content in ash tree flower polysaccharide |
| CN1271403C (en) * | 2004-07-16 | 2006-08-23 | 上海中医药大学 | Polysaccharide quantitative detecting method and system |
| JP2006288303A (en) * | 2005-04-12 | 2006-10-26 | Sagami Keisoku Kk | Method for purification of riboflavin glycoside and method for analysis thereof |
| CN100485387C (en) * | 2006-11-07 | 2009-05-06 | 王冕 | Method for detecting lentinan molecular weight and molecular weight distribution |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936901A (en) * | 2009-06-30 | 2011-01-05 | 怀亚特技术公司 | The characterizing method of macromolecular reversible association in the solution of high concentration |
| CN101936901B (en) * | 2009-06-30 | 2014-09-17 | 怀亚特技术公司 | Method for characterizing reversible association of macromolecules at high concentration |
| CN114605566A (en) * | 2022-03-14 | 2022-06-10 | 中国科学院长春应用化学研究所 | Method for extracting aloe gel macromolecule active ingredient and application thereof |
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