CN101716283A - Method for constructing dendrobium officinale polysaccharide fingerprinting - Google Patents
Method for constructing dendrobium officinale polysaccharide fingerprinting Download PDFInfo
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Abstract
The invention relates to a method for constructing dendrobium officinale polysaccharide fingerprinting, which concretely comprises the following seven operation steps: extracting dendrobium officinale polysaccharide, carrying out zymohydrolysis on the dendrobium officinale polysaccharide, deriving a dendrobium officinale polysaccharide zymolyte, constructing an oligosaccharide scale, deriving oligosaccharide of different polymerization degrees and constructing the dendrobium polysaccharide fingerprinting of different producing areas and different varieties. The method of extracting the dendrobium officinale polysaccharide of different producing areas and different varieties, the method of carrying out zymohydrolysis on the polysaccharide and the method of deriving a polysaccharide zymohydrolysis product are the same as the steps (1), (2) and (3) and can be respectively operated simultaneously. The method for constructing the dendrobium officinale polysaccharide fingerprinting by utilizing a fluorescent auxiliary sugar electrophoresis technology has the advantages of simple and sensitive operation, high efficiency and the like, is a novel method for identifying and evaluating traditional Chinese medicinal materials and makes up for the defect that the traditional method for identifying and evaluating the traditional Chinese medicinal materials is lack of the relevant information of medicinal active substances.
Description
Technical field
The invention belongs to the Chinese herbal medicine resource field, be specifically related to the construction method of dendrobium officinale polysaccharide fingerprinting.
Background technology
Herba Dendrobii is one of famous and precious medicinal plants of clearly putting down in writing of successive dynasties book on Chinese herbal medicine and Chinese Pharmacopoeia, and polysaccharide is main matter basis of its performance pharmacotoxicological effect.Modern pharmacology studies show that Herba Dendrobii has various biological effects such as resisting fatigue, antioxidation, enhancing human body immunity power, antitumor, antiplatelet aggregation, blood sugar lowering.In recent years Herba Dendrobii exploitation has been become plurality of health care functions food,, cost an arm and a leg as Herba Dendrobii Herba Dendrobii extract crystalline substance etc.Since Herba Dendrobii to growing environment require special, the self-reproduction coefficient is low and excessively the excavating of people, and causes its wild resource to be on the verge of exhaustion.The Herba Dendrobii of selling on the market is artificial growth kind or kind of poor quality mostly, and it is very complicated to originate, the good and bad quality that directly influences medical material of provenance.Herba Dendrobii resource chaotic unclear seriously limited people it carried out rational development and utilization.Current Idioplasm identification to Herba Dendrobii mainly adopts two kinds of means of molecular fingerprint of morphology and PCR-based technology.The Herba Dendrobii form of separate sources is quite similar, especially is processed into after the Herba Dendrobii extract more difficult distinguishing.Though the molecular fingerprint of PCR-based technology is being brought into play very important effect aspect the natural resources of Chinese medicinal materials evaluation, it lacks the index of correlation of bioactive ingredients content, can not estimate quality of medicinal material.In addition, molecular fingerprint is identified complicated operating process, required instrument, drug price costliness, and this has also seriously limited its promotion and application.Therefore, being badly in need of a kind of new Herba Dendrobii resource of exploitation identifies and the quality evaluation technology.
Fluorescence assisted carbohydrate electrophoresis technology (Fluorophore-assisted carbohydrate electrophoresis, FACE) be a kind of sugared electrophoretic techniques that developed recently gets up, its principle is to utilize fluorescent marker and glycan molecule to carry out nucleophilic addition, make electric charge certain on the sugar subband, by polyacrylamide gel electrophoresis (PAGE) or high performance capillary electrophoresis (HPCE) the derivatization product is separated again.Simple to operate, sensitive, efficient advantages of higher that this kind method has.Polysaccharide is the macromolecular compound that is polymerized by glycosidic bond by monosaccharide, and polysaccharide structures (monosaccharide composition, connected mode, degree of branching, substituent group etc.) is the basis of its performance pharmacological activity.
Summary of the invention
Identify the problem of complicated operation, expense costliness in order to solve existing Herba Dendrobii, the present invention utilizes the fluorescence assisted carbohydrate electrophoresis technology that a kind of construction method of dendrobium officinale polysaccharide fingerprinting is provided.
The concrete operational approach of the present invention is as follows:
The construction method of dendrobium officinale polysaccharide fingerprinting comprises following operating procedure:
(1) extracting dendrobium officinale polysaccharide
Get the exsiccant Herba Dendrobii stem of 10.0g powder, add the dual distilled water of 100mL, extract 4h, repeat 3 times in 70 ℃ of water-baths; Collect lixiviating solution, be evaporated to 50mL, add 200mL 95% ethanol (analytical pure), under 4 ℃, leave standstill 24h, centrifugal (15min, 12000 * g) collecting precipitation things; The precipitate of collecting is waved most ethanol, uses the dual dissolved in distilled water of 10mL, and removes the protein reagent deproteinization with 5mL, and centrifugal collection supernatant gets the 1g Dendrobium officinale polysaccharide through lyophilization;
(2) enzymolysis of Dendrobium officinale polysaccharide
Get the above-mentioned Dendrobium officinale polysaccharide of 50mg is mixed with 5mg/mL with the dual distilled water of 10mL Dendrobium officinale polysaccharide solution; Get Dendrobium officinale polysaccharide solution 10 μ L, and to add concentration be the beta-mannase enzymatic solution 15 μ L of 5mg/mL, vortex mixing, centrifugal 10 seconds, 50 ℃ of hydrolysis 24h; Boiling water bath 10min cessation reaction, the centrifugal 10min of 5000 * g, 25 μ L supernatant of collection are the Dendrobium officinale polysaccharide enzymolysis solution;
(3) derivatization of Dendrobium officinale polysaccharide zymolyte
1. the Dendrobium officinale polysaccharide enzymolysis solution with above-mentioned 25 μ L moves in the clean centrifuge tube of 1.5mL 45 ℃ of following water bath method solution;
2. adding 5 μ L concentration is the 8-amino naphthalenes-1,3 of 0.2mol/L, 6-trisulfonic acid solution, and mixing gets 30 μ L mixed liquor A;
3. adding 5 μ L concentration in mixed liquor A is the sodium cyanoborohydride solution of 1mol/L, and the vortex mixing, centrifugal 10 seconds, and make centrifugal liquid in pipe all sink to the pipe end, reacted 16 hours down in 37 ℃, get 35 μ L reaction mixture B;
4. reaction mixture B is in 45 ℃ of following water bath methods, and water bath method adds 20% (v/v) glycerol, and gained solution is the 8-amino naphthalenes-1,3 of Dendrobium officinale polysaccharide zymolyte, and 6-trisulfonic acid derivatization product glycerite is used for the polyacrylamide gel electrophoresis analysis;
(4) structure of oligosaccharide scale
Get the glucosan that the 0.2g molecular weight is 20000Da, adding 1.5mL concentration is the dissolving with hydrochloric acid of 0.1mol/L, and reacts 1.5h down in 50 ℃; The reactant liquor water bath method adds the dual distilled water of 0.5mL again, 45 ℃ of water bath methods, triplicate (triplicate is to remove residual hydrochloric acid); Evaporate to dryness gained solid matter is the oligosaccharide mixture of different polymerization degree; The oligosaccharide mixture that adds the dual dissolved in distilled water different polymerization degree of 1.0mL again, the centrifugal 10min of 5000 * g, the 1.0mL supernatant of collection is the oligosaccharide mixed solution of different polymerization degree;
(5) derivatization of different polymerization degree oligosaccharide
1. get the different polymerization degree oligosaccharide mixed liquor of above-mentioned 25 μ L, move in the clean centrifuge tube of 1.5mL 45 ℃ of following water bath methods;
2. adding 5 μ L concentration is the 8-amino naphthalenes-1,3 of 0.2mol/L, 6-trisulfonic acid solution, and mixing gets 30 μ L mixed liquor C;
3. adding 5 μ L concentration in mixed liquor C is the sodium cyanoborohydride solution of 1mol/L, and the vortex mixing, centrifugal 10 seconds, and make centrifugal liquid in pipe all sink to the pipe end, in 37 ℃ of reactions of temperature 16 hours, get 35 μ L reaction mixture D;
4. reaction mixture D is in 45 ℃ of following water bath methods, add 20% (v/v) glycerol in the water bath method thing, gained solution is the 8-amino naphthalenes-1,3 of different polymerization degree oligosaccharide, 6-trisulfonic acid derivatization product glycerol mixed solution is used for the polyacrylamide gel electrophoresis analysis;
The enzyme solution of the Dendrobium officinale polysaccharide in the different places of production and the extracting dendrobium officinale polysaccharide method of different cultivars, polysaccharide and the derivatization method of polyase hydrolysis products are with above-mentioned (1)-(3) step, can operate respectively simultaneously, for following step (6) and step (7) are prepared.
(6) structure of different place of production dendrobium officinale polysaccharide fingerprinting
1. be Tris-borate buffer of 8.2 with 0.1mol/L, pH value, place refrigerator to be chilled to 4 ℃ in advance, the pre-cooling electrode buffer;
2. the vertical slab electrophoresis groove of two blocks of offset plates being packed into, the distance between the offset plate is no more than 1mm;
3. prepare 12.021mL separation gel solution
Pipette the gel storage liquid of 9.0mL concentration 40% (w/v) in order respectively, 3mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2,15 μ L concentration are the Ammonium persulfate. of 10% (w/v), and 6 μ L tetramethylethylenediamines are mixed rapidly;
4. get 9mL separation gel solution, and be injected between two blocks of offset plates;
5. add the water saturation aqueous isopropanol above the separation gel solution between two blocks of offset plates, make the separation gel upper end cover the high water saturation aqueous isopropanol of one deck 0.5cm～1cm, leave standstill 2h, gel polymerisation is complete;
6. prepare 4.025mL and concentrate sol solution
Pipette 0.8mL 40% (w/v) gel storage liquid in order, 1mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2, the dual distilled water of 2.2mL, and 20 μ L concentration are the Ammonium persulfate. of 10% (w/v), 5.0 μ L tetramethylethylenediamine is mixed rapidly;
7. with the water saturation aqueous isopropanol on filter paper exhaustion separation gel upper strata, add concentrated sol solution; Insert 12 hole combs of 1.0mm thickness, be used to make the load sample hole;
8. wait to concentrate the sol solution polymerization and finish, take out comb vertically upward, wash the load sample hole 3 times, remove unpolymerized acrylamide with electrode buffer; In electrophoresis tank, add 800mL pre-cooling electrode buffer.
9. with the 8-amino naphthalenes-1 of different places of production Dendrobium officinale polysaccharide zymolyte, 3, the 8-amino naphthalenes-1 of 6-trisulfonic acid derivatization product glycerite, different polymerization degree oligosaccharide, 3,6-trisulfonic acid derivatization product glycerol mixed solution and spike indicator are injected different load sample holes respectively with microsyringe, and every hole sample size is 4.0 μ L;
10. electrophoresis tank is placed 4 ℃ of refrigerators, energized, beginning 30min current stabilization 8.0mA, current stabilization 15.0mA when treating that the spike indicator migrates to the separation gel centre position, stops electrophoresis subsequently;
(11) take out gel, place on the ultraviolet reflectance projectoscope, observe under the 360nm excitation wavelength and take pictures, the gained photo is dendrobium officinale polysaccharide fingerprinting;
(7) structure of different cultivars dendrobium polysaccharide finger printing
The structure of the same step of operational approach (6) dendrobium officinale polysaccharide fingerprinting.
The compound method of described beta-mannase enzymatic solution: take by weighing the 5mg 'beta '-mannase, it is dissolved in 1mL concentration is that 0.1mol/L, pH value are 5.0 ammonium acetate solution.
The 8-amino naphthalenes-1,3 of described 0.2mol/L, the compound method of 6-trisulfonic acid solution: take by weighing 8.1mg 8-amino naphthalenes-1,3, the 6-trisulfonic acid, adding concentration is the acetic acid 100 μ L of 15% (v/v), 60 ℃ of dissolvings.
The compound method of the sodium cyanoborohydride solution of described 1mol/L: take by weighing the 6.3mg sodium cyanoborohydride, be dissolved in the dimethyl sulfoxine of 100 μ L.
The 8-amino naphthalenes-1,3 of described 0.2mol/L, the compound method of 6-trisulfonic acid solution: take by weighing 8.1mg 8-amino naphthalenes-1,3, the 6-trisulfonic acid, adding concentration is the acetic acid 100 μ L of 15% (v/v), 60 ℃ of dissolvings.
The compound method of the sodium cyanoborohydride solution of described 1mol/L: take by weighing the 6.3mg sodium cyanoborohydride, be dissolved in the dimethyl sulfoxine of 100 μ L.
The collocation method of described 40% gel storage liquid: take by weighing the two fork of 38.7g acrylamide and 1.3g acrylamide, add dual dissolved in distilled water and be settled to 100.0mL.
The compound method of described spike indicator: taking by weighing the 0.1mg bromophenol blue, is that 0.2mol/L, pH value are 8.2 Tris-borate buffer and the dissolving of 1mL glycerol mixed liquor with 2mL concentration, and the dual distilled water of reuse is settled to 10mL.
The present invention utilizes narrow spectrum glycoside hydrolase, polysaccharide hydrolysis is become the oligosaccharide molecular of different polymerization degree.The polysaccharide of different structure, the hydrolysis site of glycosidase will inevitably there are differences, thereby forms different narrow spectrum oligose fragment, utilizes fluorescence assisted carbohydrate electrophoresis to separate, and finally can obtain the enzyme action finger printing of different polysaccharide molecules.Polysaccharide composition, content and the architectural difference thereof of separate sources are bigger, even the medical material in same kind, the different places of production, its enzyme action finger printing also exists than big-difference.Polysaccharide is the main bioactive substance of Herba Dendrobii, adopts the FACE technology to set up the dendrobium polysaccharide finger printing and can effectively identify and quality evaluation the separate sources Herba Dendrobii.
Useful technique effect of the present invention is embodied in the following aspects:
(1) dendrobium officinale polysaccharide fingerprinting (Fig. 1) based on the fluorescence assisted carbohydrate electrophoresis technique construction can identify the Herba Dendrobii that produce in Anhui, Fujian, Yunnan, Guangdong and Guangxi; Herba Dendrobii is produced in Wenzhou, Zhejiang, Wild Goose and Reed Marsh Mountains, Zhejiang, Jiangxi, Hunan, Fuyang, Zhejiang and Yunnan can be classified as a class.
(2) the different cultivars dendrobium polysaccharide finger printing (Fig. 2) based on the fluorescence assisted carbohydrate electrophoresis technique construction can identify Herba Dendrobii, gold 7 different cultivars such as dry measure used in former times, Dendrobium Moniliforme, purple dendrobium, pasture and water Herba Dendrobii, wide arthrolith dry measure used in former times and tack Herba Dendrobii suddenly.
(3) the fluorescence assisted carbohydrate electrophoresis technology has simple to operate, sensitive, efficient advantages of higher, is a kind of new evaluation and the method for estimating Chinese crude drug, and it has remedied existing evaluation and has estimated the deficiency that lacks the active medicinal matter relevant information in the method for Chinese crude drug.
Description of drawings
Fig. 1 is the dendrobium officinale polysaccharide fingerprinting figure based on the fluorescence assisted carbohydrate electrophoresis technique construction,
Fig. 2 is the different cultivars dendrobium polysaccharide fingerprint image spectrogram based on the fluorescence assisted carbohydrate electrophoresis technique construction.
The specific embodiment
Below in conjunction with embodiment, the present invention is done to describe further.
Embodiment:
With Wenzhou, Zhejiang Herba Dendrobii is example, and the concrete operations step is as follows:
(1) Wenzhou, Zhejiang extracting dendrobium officinale polysaccharide:
Get Wenzhou, the exsiccant Zhejiang of 10.0g Herba Dendrobii stem powder, add the dual distilled water of 100mL, extract 4h, repeat 3 times in 70 ℃ of water-baths.Collect lixiviating solution, be evaporated to 50mL, add 200mL 95% ethanol (analytical pure), under 4 ℃, leave standstill 24h, centrifugal (15min, 12000 * g) collecting precipitation things.The precipitation of collecting is waved most ethanol, with the dual dissolved in distilled water of 10mL, and with 5mL remove protein reagent (chloroform: n-butyl alcohol=4: 1, v/v) deproteinization, centrifugal collection supernatant gets Wenzhou, 1g Zhejiang Dendrobium officinale polysaccharide through lyophilization.
(2) enzymolysis of Wenzhou, Zhejiang Dendrobium officinale polysaccharide:
Get Wenzhou, the above-mentioned Zhejiang of 50mg Dendrobium officinale polysaccharide is mixed with 5mg/mL with the dual distilled water of 10mL Dendrobium officinale polysaccharide solution, get this solution 10 μ L, and adding concentration is the beta-mannase enzymatic solution 15 μ L (preparation of beta-mannase enzymatic solution: take by weighing the 5mg 'beta '-mannase of 5mg/mL, it is dissolved in 1mL concentration is that 0.1mol/L, pH value are 5.0 ammonium acetate solution), the vortex mixing, centrifugal 10 seconds, 50 ℃ of hydrolysis 24h.Boiling water bath 10min cessation reaction, the centrifugal 10min of 5000 * g, the supernatant of collection are Wenzhou, Zhejiang Dendrobium officinale polysaccharide enzymolysis solution (25 μ L).
(3) derivatization of Wenzhou, Zhejiang Dendrobium officinale polysaccharide zymolyte:
1. Wenzhou, the Zhejiang Dendrobium officinale polysaccharide enzymolysis solution with above-mentioned 25 μ L moves in the clean centrifuge tube of 1.5mL 45 ℃ of following water bath method solution.
2. adding 5 μ L concentration is the 8-amino naphthalenes-1 of 0.2mol/L, 3,6-trisulfonic acid solution [the 8-amino naphthalenes-1,3 of 0.2mol/L, the preparation of 6-trisulfonic acid solution: take by weighing 8.1mg 8-amino naphthalenes-1,3, the 6-trisulfonic acid, adding concentration is the acetic acid 100 μ L of 15% (v/v), 60 ℃ of dissolvings], mixing gets mixed liquor A (30 μ L).
3. adding 5 μ L concentration in mixed liquor A is the sodium cyanoborohydride solution (preparation of the sodium cyanoborohydride solution of 1mol/L: take by weighing the 6.3mg sodium cyanoborohydride of 1mol/L, be dissolved in the dimethyl sulfoxine of 100 μ L), and vortex mixing, centrifugal 10 seconds, make centrifugal liquid in pipe all sink to the pipe end, reacted 16 hours down in 37 ℃, get reaction mixture B (35 μ L).
4. reaction mixture B behind the water bath method, adds 20% (v/v) glycerol to it in 45 ℃ of following water bath methods, gained solution is the 8-amino naphthalenes-1 of Wenzhou, Zhejiang Dendrobium officinale polysaccharide zymolyte, 3,6-trisulfonic acid derivatization product glycerite is used for the polyacrylamide gel electrophoresis analysis.
(4) derivatization method of the enzyme solution of the extracting method of other place of production Dendrobium officinale polysaccharide and other kind dendrobium polysaccharide, polysaccharide and polyase hydrolysis products is identical with above-mentioned (1)-(3) step process method, can operate respectively simultaneously.
(5) structure of oligosaccharide scale:
Get the glucosan that the 0.2g molecular weight is 20000Da, adding 1.5mL concentration is the dissolving with hydrochloric acid of 0.1mol/L, and reacts 1.5h down in 50 ℃.The reactant liquor water bath method adds the dual distilled water of 0.5mL again, 45 ℃ of water bath methods (triplicate is to remove residual hydrochloric acid), and the residual solids material is the oligosaccharide mixture of different polymerization degree.The oligosaccharide mixture that adds the dual dissolved in distilled water different polymerization degree of 1.0mL again, the centrifugal 10min of 5000 * g, the supernatant of collection are the oligosaccharide mixed solution (1.0mL) of different polymerization degree, for following step (6) is prepared.
(6) derivatization of different polymerization degree oligosaccharide:
1. get the different polymerization degree oligosaccharide mixed liquor of above-mentioned 25 μ L, move in the clean centrifuge tube of 1.5mL 45 ℃ of following water bath method solution.
2. adding 5 μ L concentration is the 8-amino naphthalenes-1,3 of 0.2mol/L, 6-trisulfonic acid solution, and mixing gets mixed liquor C (30 μ L).
0.2mol/L 8-amino naphthalenes-1,3, the preparation of 6-trisulfonic acid solution: take by weighing 8.1mg 8-amino naphthalenes-1,3, the 6-trisulfonic acid, adding concentration is the acetic acid 100 μ L of 15% (v/v), 60 ℃ of dissolvings.
3. adding 5 μ L concentration in mixed liquor C is the sodium cyanoborohydride solution of 1mol/L, and the vortex mixing, centrifugal 10 seconds, and make centrifugal liquid in pipe all sink to the pipe end, reacted 16 hours down in 37 ℃, get 35 μ L reaction mixture D.
The preparation of the sodium cyanoborohydride solution of 1mol/L: take by weighing the 6.3mg sodium cyanoborohydride, be dissolved in the dimethyl sulfoxine of 100 μ L.
4. reaction mixture D behind the water bath method, adds 20% (v/v) glycerol to it in 45 ℃ of following water bath methods, gained solution is the 8-amino naphthalenes-1 of different polymerization degree oligosaccharide, 3,6-trisulfonic acid derivatization product glycerol mixed solution is used for the polyacrylamide gel electrophoresis analysis.
The 8-amino naphthalenes-1,3 of preparation different polymerization degree oligosaccharide, 6-trisulfonic acid derivatization product is that following step (7) and step (8) are prepared, as the molecular weight scale of polysaccharide fingerprinting.
(7) structure of dendrobium officinale polysaccharide fingerprinting
1. prepare the 1000mL electrode buffer
Electrode buffer is Tris-borate buffer of 0.1mol/L, and pH 8.2, and places refrigerator to be chilled to 4 ℃ in advance electrode buffer.
2. assemble two blocks of offset plates, the distance between the offset plate is no more than 1mm, and with its vertical slab electrophoresis groove of packing into.
3. prepare 12.021mL separation gel solution: pipetting 9.0mL concentration in order respectively is the gel storage liquid of 40% (w/v), 3mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2,15 μ L concentration are the Ammonium persulfate. of 10% (w/v), 6 μ L tetramethylethylenediamines mix rapidly.
The configuration of used 40% gel storage liquid: take by weighing the two fork of 38.7g acrylamide and 1.3g acrylamide, add dual dissolved in distilled water and be settled to 100.0mL.
4. get 9mL separation gel solution, and be injected between two blocks of offset plates, note not producing bubble.
5. add the water saturation aqueous isopropanol above the separation gel solution between two blocks of offset plates, make the separation gel upper end cover the high water saturation aqueous isopropanol of one deck 0.5cm～1cm, leave standstill 2h and wait for that gel polymerisation is complete.
6. prepare 4.025mL and concentrate sol solution: pipette 0.8mL 40% (w/v) gel storage liquid in order, 1mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2,2.2mL dual distilled water, 20 μ L concentration are the Ammonium persulfate. of 10% (w/v), 5.0 μ L tetramethylethylenediamine mixes rapidly.
7. with the aqueous isopropanol on filter paper exhaustion separation gel upper strata, pour concentrated sol solution into.Insert the 12 hole combs (being used to make the load sample hole) of 1.0mm thickness.
8. wait to concentrate the sol solution polymerization and finish, take out comb vertically upward, wash the load sample hole 3 times, remove unpolymerized acrylamide with electrode buffer.The electrode buffer that in electrophoresis tank, adds the 800mL pre-cooling.
9. with the 8-amino naphthalenes-1 of different places of production Dendrobium officinale polysaccharide zymolyte, 3, the 8-amino naphthalenes-1 of 6-trisulfonic acid derivatization product glycerite, different polymerization degree oligosaccharide, 3,6-trisulfonic acid derivatization product glycerol mixed solution and spike indicator are injected different load sample holes respectively with microsyringe, and every hole sample size is 4.0 μ L.
The preparation of used spike indicator: taking by weighing the 0.1mg bromophenol blue, is that 0.2mol/L, pH value are 8.2 Tris-borate buffer and the dissolving of 1mL glycerol mixed liquor with 2mL concentration, and the dual distilled water of reuse is settled to 10mL.
10. electrophoresis tank is placed 4 ℃ of refrigerators, energized, beginning 30min current stabilization 8.0mA, current stabilization 15.0mA when treating that the spike indicator migrates to the separation gel centre position, stops electrophoresis subsequently.
(11) take out gel, place on the ultraviolet reflectance projectoscope, observe under the 360nm excitation wavelength and take pictures, the gained photo is dendrobium officinale polysaccharide fingerprinting.
(8) structure of different cultivars dendrobium polysaccharide finger printing
1. prepare the 1000mL electrode buffer
Electrode buffer is Tris-borate buffer of 0.1mol/L, pH8.2, and place refrigerator to be chilled to 4 ℃ in advance the electrode buffer.
2. assemble two blocks of offset plates, the distance between the offset plate is no more than 1mm, and with its vertical slab electrophoresis groove of packing into.
3. prepare 12.021mL separation gel solution: pipetting 9.0mL concentration in order respectively is the gel storage liquid of 40% (w/v), 3mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2,15 μ L concentration are the Ammonium persulfate. of 10% (w/v), 6 μ L tetramethylethylenediamines mix rapidly.
The configuration of used 40% gel storage liquid: take by weighing the two fork of 38.7g acrylamide and 1.3g acrylamide, add dual dissolved in distilled water and be settled to 100.0mL.
4. get 9mL separation gel solution, and be injected between two blocks of offset plates, note not producing bubble.
5. add the water saturation aqueous isopropanol above the separation gel solution between two blocks of offset plates, make the separation gel upper end cover the high water saturation aqueous isopropanol of one deck 0.5cm～1cm, leave standstill 2h and wait for that gel polymerisation is complete.
6. prepare 4.025mL and concentrate sol solution: pipette 0.8mL 40% (w/v) gel storage liquid in order, 1mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2,2.2mL dual distilled water, 20 μ L concentration are the Ammonium persulfate. of 10% (w/V), 5.0 μ L tetramethylethylenediamine mixes rapidly.
7. with the aqueous isopropanol on filter paper exhaustion separation gel upper strata, pour concentrated sol solution into.Insert the 12 hole combs (being used to make the load sample hole) of 1.0mm thickness.
8. wait to concentrate the sol solution polymerization and finish, take out comb vertically upward, wash the load sample hole 3 times, remove unpolymerized acrylamide with electrode buffer.The electrode buffer that in electrophoresis tank, adds the 800mL pre-cooling.
9. with the 8-amino naphthalenes-1 of different cultivars dendrobium polysaccharide zymolyte, 3, the 8-amino naphthalenes-1 of 6-trisulfonic acid derivatization product glycerite, different polymerization degree oligosaccharide, 3,6-trisulfonic acid derivatization product glycerol mixed solution and spike indicator are injected different load sample holes respectively with microsyringe, and every hole sample size is 4.0 μ L.
The preparation of used spike indicator: taking by weighing the 0.1mg bromophenol blue, is that 0.2mol/L, pH value are 8.2 Tris-borate buffer and the dissolving of 1mL glycerol mixed liquor with 2mL concentration, and the dual distilled water of reuse is settled to 10mL.
10. electrophoresis tank is placed 4 ℃ of refrigerators, energized, beginning 30min current stabilization 8.0mA, current stabilization 15.0mA when treating that the spike indicator migrates to the separation gel centre position, stops electrophoresis subsequently.
(11) take out gel, place on the ultraviolet reflectance projectoscope, observe under the 360nm excitation wavelength and take pictures, the gained photo is different cultivars dendrobium polysaccharide finger printing.
Referring to Fig. 1, can identify the Herba Dendrobii of Anhui, Fujian, Yunnan, Guangdong and Guangxi product based on the dendrobium officinale polysaccharide fingerprinting of fluorescence assisted carbohydrate electrophoresis technique construction; Herba Dendrobii is produced in Wenzhou, Zhejiang, Wild Goose and Reed Marsh Mountains, Zhejiang, Jiangxi, Hunan, Fuyang, Zhejiang and Yunnan can be classified as a class.Among Fig. 1,1 represents the different polymerization degree oligosaccharide scale; 2 represent glucose; 3 represent Wenzhou, Zhejiang Herba Dendrobii; 4 represent Wild Goose and Reed Marsh Mountains, Zhejiang Herba Dendrobii; 5 represent the Guangxi Herba Dendrobii; 6 represent the Jiangxi Herba Dendrobii; 7 represent the Hunan Herba Dendrobii; 8 represent the Guangdong Herba Dendrobii; 9 represent the Fujian Herba Dendrobii; 10 represent the Fuyang, Zhejiang Herba Dendrobii; 11 represent the Yunnan Herba Dendrobii; 12 represent the Anhui Herba Dendrobii.
Referring to Fig. 2, can identify Herba Dendrobii, gold 7 different cultivars such as dry measure used in former times, Dendrobium Moniliforme, purple dendrobium, pasture and water Herba Dendrobii, wide arthrolith dry measure used in former times and tack Herba Dendrobii suddenly based on the different cultivars dendrobium polysaccharide finger printing of fluorescence assisted carbohydrate electrophoresis technique construction.Among Fig. 2,1 represents the different polymerization degree oligosaccharide scale; 2 represent glucose; 3 represent Herba Dendrobii; 4 represent the Jin Huo dry measure used in former times; 5 represent Dendrobium Moniliforme; 6 represent purple dendrobium; 7 represent the pasture and water Herba Dendrobii; The wide arthrolith dry measure used in former times of 8 representatives; 9 represent the tack Herba Dendrobii.
Claims (8)
1. the construction method of dendrobium officinale polysaccharide fingerprinting is characterized in that comprising following operating procedure:
(1) extracting dendrobium officinale polysaccharide
Get the exsiccant Herba Dendrobii stem of 10.0g powder, add the dual distilled water of 100mL, extract 4h, repeat 3 times in 70 ℃ of water-baths; Collect lixiviating solution, be evaporated to 50mL, add 200mL 95% ethanol (analytical pure), under 4 ℃, leave standstill 24h, centrifugal (15min, 12000 * g) collecting precipitation things; The precipitate of collecting is waved most ethanol, uses the dual dissolved in distilled water of 10mL, and removes the protein reagent deproteinization with 5mL, and centrifugal collection supernatant gets the 1g Dendrobium officinale polysaccharide through lyophilization;
(2) enzymolysis of Dendrobium officinale polysaccharide
Get the above-mentioned Dendrobium officinale polysaccharide of 50mg is mixed with 5mg/mL with the dual distilled water of 10mL Dendrobium officinale polysaccharide solution; Get Dendrobium officinale polysaccharide solution 10 μ L, and to add concentration be the beta-mannase enzymatic solution 15 μ L of 5mg/mL, vortex mixing, centrifugal 10 seconds, 50 ℃ of hydrolysis 24h; Boiling water bath 10min cessation reaction, the centrifugal 10min of 5000 * g, 25 μ L supernatant of collection are the Dendrobium officinale polysaccharide enzymolysis solution;
(3) derivatization of Dendrobium officinale polysaccharide zymolyte
1. the Dendrobium officinale polysaccharide enzymolysis solution with above-mentioned 25 μ L moves in the clean centrifuge tube of 1.5mL 45 ℃ of following water bath method solution;
2. adding 5 μ L concentration is the 8-amino naphthalenes-1,3 of 0.2mol/L, 6-trisulfonic acid solution, and mixing gets 30 μ L mixed liquor A;
3. adding 5 μ L concentration in mixed liquor A is the sodium cyanoborohydride solution of 1mol/L, and the vortex mixing, centrifugal 10 seconds, and make centrifugal liquid in pipe all sink to the pipe end, reacted 16 hours down in 37 ℃, get 35 μ L reaction mixture B;
4. reaction mixture B is in 45 ℃ of following water bath methods, and water bath method adds 20% (v/v) glycerol, and gained solution is the 8-amino naphthalenes-1,3 of Dendrobium officinale polysaccharide zymolyte, and 6-trisulfonic acid derivatization product glycerite is used for the polyacrylamide gel electrophoresis analysis;
(4) structure of oligosaccharide scale
Get the glucosan that the 0.2g molecular weight is 20000Da, adding 1.5mL concentration is the dissolving with hydrochloric acid of 0.1mol/L, and reacts 1.5h down in 50 ℃; The reactant liquor water bath method adds the dual distilled water of 0.5mL again, 45 ℃ of water bath methods, triplicate (triplicate is to remove residual hydrochloric acid); Evaporate to dryness gained solid matter is the oligosaccharide mixture of different polymerization degree; The oligosaccharide mixture that adds the dual dissolved in distilled water different polymerization degree of 1.0mL again, the centrifugal 10min of 5000 * g, the 1.0mL supernatant of collection is the oligosaccharide mixed solution of different polymerization degree;
(5) derivatization of different polymerization degree oligosaccharide
1. get the different polymerization degree oligosaccharide mixed liquor of above-mentioned 25 μ L, move in the clean centrifuge tube of 1.5mL 45 ℃ of following water bath methods;
2. adding 5 μ L concentration is the 8-amino naphthalenes-1,3 of 0.2mol/L, 6-trisulfonic acid solution, and mixing gets 30 μ L mixed liquor C;
3. adding 5 μ L concentration in mixed liquor C is the sodium cyanoborohydride solution of 1mol/L, and the vortex mixing, centrifugal 10 seconds, and make centrifugal liquid in pipe all sink to the pipe end, in 37 ℃ of reactions of temperature 16 hours, get 35 μ L reaction mixture D;
4. reaction mixture D is in 45 ℃ of following water bath methods, add 20% (v/v) glycerol in the water bath method thing, gained solution is the 8-amino naphthalenes-1,3 of different polymerization degree oligosaccharide, 6-trisulfonic acid derivatization product glycerol mixed solution is used for the polyacrylamide gel electrophoresis analysis;
The enzyme solution of the Dendrobium officinale polysaccharide in the different places of production and the extracting dendrobium officinale polysaccharide method of different cultivars, polysaccharide and the derivatization method of polyase hydrolysis products are with above-mentioned (1)-(3) step, can operate respectively simultaneously, for following step (6) and step (7) are prepared;
(6) structure of different place of production dendrobium officinale polysaccharide fingerprinting
1. be Tris-borate buffer of 8.2 with 0.1mol/L, pH value, place refrigerator to be chilled to 4 ℃ in advance, the pre-cooling electrode buffer;
2. the vertical slab electrophoresis groove of two blocks of offset plates being packed into, the distance between the offset plate is no more than 1mm;
3. prepare 12.021mL separation gel solution
Pipette the gel storage liquid of 9.0mL concentration 40% (w/v) in order respectively, 3mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2,15 μ L concentration are the Ammonium persulfate. of 10% (w/v), and 6 μ L tetramethylethylenediamines are mixed rapidly;
4. get 9mL separation gel solution, and be injected between two blocks of offset plates;
5. add the water saturation aqueous isopropanol above the separation gel solution between two blocks of offset plates, make the separation gel upper end cover the high water saturation aqueous isopropanol of one deck 0.5cm～1cm, leave standstill 2h, gel polymerisation is complete;
6. prepare 4.025mL and concentrate sol solution
Pipette 0.8mL 40% (w/v) gel storage liquid in order, 1mL concentration is that 0.8mol/L, pH value are Tris-borate buffer of 8.2, the dual distilled water of 2.2mL, and 20 μ L concentration are the Ammonium persulfate. of 10% (w/v), 5.0 μ L tetramethylethylenediamine is mixed rapidly;
7. with the water saturation aqueous isopropanol on filter paper exhaustion separation gel upper strata, add concentrated sol solution; Insert 12 hole combs of 1.0mm thickness, be used to make the load sample hole;
8. wait to concentrate the sol solution polymerization and finish, take out comb vertically upward, wash the load sample hole 3 times, remove unpolymerized acrylamide with electrode buffer; In electrophoresis tank, add 800mL pre-cooling electrode buffer.
9. with the 8-amino naphthalenes-1 of different places of production Dendrobium officinale polysaccharide zymolyte, 3, the 8-amino naphthalenes-1 of 6-trisulfonic acid derivatization product glycerite, different polymerization degree oligosaccharide, 3,6-trisulfonic acid derivatization product glycerol mixed solution and spike indicator are injected different load sample holes respectively with microsyringe, and every hole sample size is 4.0 μ L;
10. electrophoresis tank is placed 4 ℃ of refrigerators, energized, beginning 30min current stabilization 8.0mA, current stabilization 15.0mA when treating that the spike indicator migrates to the separation gel centre position, stops electrophoresis subsequently;
(11) take out gel, place on the ultraviolet reflectance projectoscope, observe under the 360nm excitation wavelength and take pictures, the gained photo is dendrobium officinale polysaccharide fingerprinting;
(7) structure of different cultivars dendrobium polysaccharide finger printing
The structure of the same step of operational approach (6) dendrobium officinale polysaccharide fingerprinting.
2. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1, it is characterized in that: the compound method of described beta-mannase enzymatic solution: take by weighing the 5mg 'beta '-mannase, it is dissolved in 1mL concentration is that 0.1mol/L, pH value are 5.0 ammonium acetate solution.
3. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1, it is characterized in that: the 8-amino naphthalenes-1 of described 0.2mol/L, 3, the compound method of 6-trisulfonic acid solution: take by weighing 8.1mg 8-amino naphthalenes-1,3, the 6-trisulfonic acid, adding concentration is the acetic acid 100 μ L of 15% (v/v), 60 ℃ of dissolvings.
4. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1 is characterized in that: the compound method of the sodium cyanoborohydride solution of described 1mol/L: take by weighing the 6.3mg sodium cyanoborohydride, be dissolved in the dimethyl sulfoxine of 100 μ L.
5. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1, it is characterized in that: the 8-amino naphthalenes-1 of described 0.2mol/L, 3, the compound method of 6-trisulfonic acid solution: take by weighing 8.1mg 8-amino naphthalenes-1,3, the 6-trisulfonic acid, adding concentration is the acetic acid 100 μ L of 15% (v/v), 60 ℃ of dissolvings.
6. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1 is characterized in that: the compound method of the sodium cyanoborohydride solution of described 1mol/L: take by weighing the 6.3mg sodium cyanoborohydride, be dissolved in the dimethyl sulfoxine of 100 μ L.
7. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1, it is characterized in that: the collocation method of described 40% gel storage liquid: take by weighing the two fork of 38.7g acrylamide and 1.3g acrylamide, add dual dissolved in distilled water and be settled to 100.0mL.
8. the construction method of dendrobium officinale polysaccharide fingerprinting according to claim 1, it is characterized in that: the compound method of described spike indicator: take by weighing the 0.1mg bromophenol blue, with 2mL concentration is that 0.2mol/L, pH value are 8.2 Tris-borate buffer and the dissolving of 1mL glycerol mixed liquor, and the dual distilled water of reuse is settled to 10mL.
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