CN101555290B - Method for preparing radix astragali homopolysaccharide - Google Patents
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Abstract
The invention provides a method for preparing radix astragali homopolysaccharide in the technical field of Chinese medicine. The method comprises the following steps that: radix astragali coarse powder is taken, refluxed and degreased, filtered, refluxed and extracted with water, concentrated, precipitated, centrifuged or filtered to collect precipitate, and the precipitate is washed and dried in vacuum to obtain radix astragali coarse polysaccharide; aqueous solution of the radix astragali coarse polysaccharide is enzymatically hydrolyzed with pepsin, boiled and centrifuged to obtain supernatant fluid; a Sevage agent is added to the supernatant fluid for shaking and extraction so as to collect upper layer aqueous phase solution; diethylaminoethyl cellulose is added to be filtered, concentrated, dialyzed, precipitated, centrifuged or filtered, washed and dried to obtain the refined radix astragali homopolysaccharide; the refined radix astragali homopolysaccharide is separated and purified through column chromatography twice, and eluted with water, the eluant is collected, concentrated, refrigerated and dried to obtain the radix astragali homopolysaccharide. The average molecular weight of the radix astragali polysaccharide obtained in the invention is about 9000 and the radix astragali homopolysaccharide only contains glucose monosaccharide unit after a complete acid hydrolysis.
Description
Technical field
The present invention relates to a kind of preparation method of technical field of traditional Chinese medicines, particularly a kind of preparation method of radix astragali homopolysaccharide.
Background technology
Polysaccharide is a class natural high moleculer eompound, is the polymer that is linked together by the glucoside key by aldose or ketose.Since finding that polysaccharide and saccharide complex participate in the adjusting of various biological phenomenas of cell the seventies in last century, people find that constantly polysaccharide has multiple biological activitys such as antitumor, immunomodulatory, hypoglycemic, antiviral, reducing blood-fat, anticoagulation.And because polysaccharide does not have obvious toxic and side effects, utilization of polysaccharide The biological resources development and research are become increasingly active, become the research focus of natural drug, biological chemistry, life science.
The Radix Astragali is the dry root of leguminous plants Radix Astragali Astragalus membranaceus (Fisch.) Bge. or Radix Astagali Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge) Hsiao, plain being celebrated with " all medicines of tonifying Qi ", is a kind of Chinese medicine of setting upright commonly used.Sweet, the tepor of nature and flavor is returned spleen, lung channel, has effects such as invigorating QI to consolidate the body surface resistance, diuresis holder poison, apocenosis, expelling pus and promoting granulation.The Radix Astragali contains multiple biologically active substances such as polysaccharide, protein, alkaloid, amino acid, flavonoid, glycoside, trace element, astragalus polysaccharides (Astragalus polysaccharides wherein, APS) be one of main bioactive ingredients of the Radix Astragali, have immunomodulatory, hypoglycemic, antitumor, promote effects such as marrow hemopoietic stem cells propagation and two-ways regulation blood sugar.
Find that through literature search Chinese patent CN101020721A has announced a kind of method for preparing the thick astragalus polysaccharides of high purity, and is centrifugal after the constant temperature microwave extraction to prior art, the supernatant liquor activated carbon decolorizing, alcohol precipitation, gel permeation chromatography are promptly; Chinese patent CN101029086A has announced that then a kind of combined utilization microwave extraction, micro-filtration, ultrafiltration and alcohol precipitation technology prepare the method for astragalus polysaccharides.Do not find further that as yet molecular weight is clear and definite, polysaccharide is formed clearly radix astragali homopolysaccharide preparation method's open source literature report.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of preparation method of radix astragali homopolysaccharide is provided.The polysaccharide molecular-weight average that method of the present invention obtains is about 9000, only contains a kind of monosaccharide unit of glucose after the acid hydrolysis fully; Method of the present invention is simply quick, and is with low cost.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
Step 1 is got radix astragali coarse powder, and with the alcohol reflux degreasing 1.5h of 3 times of amount volume fractions, wherein ethanol is that volume fraction is 95% ethanol; Filter, be incorporated as the water refluxing extraction 90min of 6～12 times of amounts of dregs of a decoction volume fraction in the dregs of a decoction, filter, repeat to extract merging filtrate 2 times;
Step 2 is got filtrate, is concentrated into 1/5～1/10 of filtrate volume, obtain concentrated solution, the adding volume fraction is 95% ethanol in concentrated solution, stirs, alcoholic acid volume fraction in solution is 50%～80%, precipitation, standing over night, centrifugal or filtration, the collecting precipitation thing, washing, 60 ℃ of vacuum-dryings promptly get Radix Astragali Crude polysaccharides;
Step 3, the Radix Astragali Crude polysaccharides that utilizes step 2 to obtain prepares 20gL
-1The Radix Astragali Crude polysaccharides aqueous solution, be that 1: 1～1: 5 ratio adds stomach en-according to Radix Astragali Crude polysaccharides and pepsic mol ratio in the Radix Astragali Crude polysaccharides aqueous solution, 37 ℃ of following enzymolysis 3～7 hours boil 10min, and are centrifugal, obtain supernatant liquor;
Step 4 is 4: 1～6: 1 a ratio in supernatant liquor and Sevage reagent volume ratio, adds Sevage reagent in supernatant liquor, and wherein Sevage reagent is specially: chloroform and propyl carbinol be by volume 4: 1 formulated; Shaking out, centrifugal and collection upper strata aqueous phase solution, repetitive operation 3～6 times;
Step 5, in the aqueous phase solution that step 4 obtains, add diethylaminoethyl cellulose, making the weight percent of diethyllaminoethyl in aqueous phase solution is 0.5%～1.5%, boil 10～20min, filter, obtain filtrate, filtrate is concentrated into 1/10 of Radix Astragali Crude polysaccharides aqueous solution volume in the step 3, concentrated solution is that 3000 dialysis tubing is dialysed with the molecular weight that dams, and dialysis 3d adds volume fraction and be 95% ethanol in the solution after dialysis, the alcoholic acid volume fraction is 80% in solution, precipitation, standing over night, centrifugal or filtration, the collecting precipitation thing, washing, 60 ℃ of vacuum-dryings promptly get the refining astragalus polysaccharide;
Step 6 is got the refining astragalus polysaccharide, ultrasonic water dissolution, and the weak negative ion-exchange chromatography of diethyllaminoethyl separates, and deionized water wash-out, flow velocity are 0.8～1.2mLmin
-1, the phenol sulfuric acid process detects, and merges the part that there is absorption at the 490nm place, concentrates, and lyophilize obtains the white solid thing;
Step 7 is dissolved in water the white solid thing that obtains in the step 6, and through the gel filtration chromatography purifies and separates, deionized water wash-out, flow velocity are 8～10mLh
-1, the HPLC-ELSD monitoring merges the longest symmetrical peak part of retention time according to the liquid phase collection of illustrative plates, concentrates, and lyophilize promptly gets radix astragali homopolysaccharide.
In step 2 and the step 5, described washing is that elder generation washes once with dehydrated alcohol, washes once with acetone again, repeats twice.
In the step 3, described stomach en-enzyme activity 〉=1200U/g.
Described column chromatography water is a deionized water.
Among the present invention, radix astragali coarse powder after the alcohol reflux degreasing, is added the water refluxing extraction, add ethanol in the water extraction concentrated solution, precipitation is filtered, collecting precipitation, and drying obtains Radix Astragali Crude polysaccharides; Crude polysaccharides is after removing albumen, depigmentation, dialysis desalting processing, through diethyllaminoethyl (DEAE) weak anion exchange resin column chromatography for separation, the deionized water wash-out, according to phenolsulfuric acid method detected result, merge elutriant, concentrated, lyophilize obtains astragalus polysaccharides, use water dissolution then, through the gel filtration chromatography separation and purification, the deionized water wash-out, with HPLC-ELSD (high performance liquid chromatography-evaporative light scattering detection) monitoring, merge the longest part that is single symmetrical peak of retention time, promptly get radix astragali homopolysaccharide.
The present invention has following beneficial effect: the polysaccharide molecular-weight average that method of the present invention obtains is about 9000, only contains a kind of monosaccharide unit of glucose after the acid hydrolysis fully; Method of the present invention is simply quick, and is with low cost.
Description of drawings
Fig. 1 is the HPLC collection of illustrative plates of resulting radix astragali homopolysaccharide.
Embodiment
Following example will the invention will be further described in conjunction with the accompanying drawings.Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Among the present invention, weak anion exchange resin can be the DEAECellulose that Britain Whatman company produces, the DEAE Sepharose FastFlow that U.S. GE Healthcare Bio-Science AB company produces, or the DEAE Sephadex A-50 of Sweden Pharmacia company production.
The gel filtration chromatography filler can be Sephacryl S-200 or the Sephadex G-150 that U.S. GE Healthcare Bio-Science AB company produces, perhaps the Superose12 PG of Sweden Pharmacia company production.
Embodiment 1
Get Milkvetch Root meal 500g, add 1500mL 95% ethanol (v/v) backflow degreasing 1.5h, filter, add 3000mL water refluxing extraction in the residue three times, each 90min.The extracting solution filtered while hot, merging filtrate is evaporated to 1800mL.Add 95% ethanol (v/v) under 4 ℃ in concentrated solution, stir, concentration of ethanol is 50% (v/v) in the regulator solution, standing over night.Centrifugal, collecting precipitation is washed once with dehydrated alcohol earlier, washes once with acetone again, repeats twice, 60 ℃ of vacuum-drying, gets pale brown toner powder Radix Astragali Crude polysaccharides 25g, and extracting yield is 5%.
Get Crude polysaccharides 20g and be mixed with 20gL
-1Aqueous solution 1000mL adds stomach en-20g, and 37 ℃ of following enzymolysis 3h boil 10min and make unnecessary proteolytic enzyme sex change settle out centrifugal removing.Adding Sevage reagent in resulting supernatant solution (chloroform: the volume ratio of propyl carbinol=4: 1) 250mL, shaking out, centrifugal, collect the upper strata aqueous phase solution, repeat this process 3 times.Adding 5g DEAE (diethyllaminoethyl) Mierocrystalline cellulose to the weight percentage of DEAE is 0.5% in the aqueous phase solution of upper strata, boils 10min, filters, and removes Mierocrystalline cellulose, and filtrate is concentrated into about 100mL.Concentrated solution is that 3000 dialysis tubing is dialysed with the molecular weight that dams, altogether 3d.Add 95% (v/v) ethanolic soln in the solution after dialysis, the alcohol concn in the regulator solution is that 80% (v/v) precipitates; Standing over night, centrifugal or filtration, the collecting precipitation thing, dehydrated alcohol, acetone replace washed twice, and 60 ℃ of vacuum-dryings get refining astragalus polysaccharide 2.4g, and yield is 6 ‰.
Get the ultrasonic water dissolution of 0.5g refining astragalus polysaccharide, carry out DEAE Sepharose Fast Flow weak anionic exchange column chromatography, deionized water wash-out, flow velocity are 0.8mLmin
-1The phenol sulfuric acid process detects, and merging the 490nm place has absorption portion, concentrates, and lyophilize obtains white powder astragalus polysaccharides 85mg.With the water-soluble after Sephacryl S-200 gel filtration chromatography purifies and separates of astragalus polysaccharides, deionized water wash-out, flow velocity are 8mLh
-1, collect water elution liquid, the HPLC-ELSD monitoring merges the longest single symmetrical peak part of retention time, concentrates, and lyophilize promptly gets white astragalus polysaccharides 34mg, and final yield is 0.41 ‰.
The gained astragalus polysaccharides is measured through HPLC-ELSD HPGPC method, and molecular-weight average is 9000; 5mL 1molL
-1H
2SO
4Behind 100 ℃ of following water-bath tube sealing hydrolysis 6h, the method inspection with ply of paper chromatography and standard substance contrast only contains D-glucose in the hydrolyzed solution, shows that this astragalus polysaccharides only is made up of glucose, is homopolysaccharide.
The measuring method that the molecular weight determination of radix astragali homopolysaccharide and complete acid hydrolysis polysaccharide are formed is:
Molecular weight determination: do typical curve as standard substance with Sigma-Aldich Dextran Standard 5000,12000,25000,50000,80000, adopt HPLC-ELSD HPGPC (efficient gel permeation chromatography) method to measure polysaccharide molecular weight, getting its molecular-weight average is 9000;
What polysaccharide was formed determines: the refining polysaccharide of 5mg is dissolved in 5mL 1molL
-1In the sulphuric acid soln, tube sealing hydrolysis 6h under 100 ℃ of water-baths; The barium carbonate neutralization, centrifugal, supernatant liquor carries out the ply of paper chromatographic analysis, with D-glucose, D-semi-lactosi, D-pectinose, L-rhamnosyl, D-wood sugar, D-seminose in contrast, (volume ratio is 5: 5: 1 to ethyl acetate-pyridine-acetic acid-water: 3) launch for developping agent, taking-up is dried, spray is with aniline-phthalic acid solution colour developing, and polysaccharide hydrolysis liquid only corresponding color dot occurs in the place consistent with D-glucose Rf value as a result, shows that this polysaccharide is made up of D-glucose.
Embodiment 2
Get Milkvetch Root meal 500g, add 1500mL 95% ethanol (v/v) backflow degreasing 1.5h, filter, add 4500mL water refluxing extraction in the residue three times, each 90min.The extracting solution filtered while hot, merging filtrate is evaporated to 1800mL.Add 95% ethanol (v/v) under 4 ℃ in concentrated solution, stir, the regulator solution determining alcohol is 65% (v/v), standing over night.Centrifugal, collecting precipitation, dehydrated alcohol, acetone replace washed twice, and 60 ℃ of vacuum-dryings get pale brown toner powder Radix Astragali Crude polysaccharides 50g, and extracting yield is 10%.
Get Crude polysaccharides 20g and be mixed with 20gL
-1Aqueous solution 1000mL adds stomach en-60g (m/m), and 37 ℃ of following enzymolysis 5h boil 10min and make unnecessary proteolytic enzyme sex change settle out centrifugal removing.Adding Sevage reagent in resulting supernatant solution (chloroform: the volume ratio of propyl carbinol=4: 1) 200mL, shaking out, centrifugal, collect the upper strata aqueous phase solution.Repeat this process 4 times.Adding 10g DEAE Mierocrystalline cellulose to concentration expressed in percentage by weight in the aqueous phase solution of upper strata is 1.0%, boils 15min, removes by filter Mierocrystalline cellulose, and filtrate is concentrated into about 100mL.Concentrated solution is that 3000 dialysis tubing is dialysed with the molecular weight that dams, altogether 3d.Add 95% (v/v) ethanolic soln in the solution after dialysis, the regulator solution determining alcohol is that 80% (v/v) precipitates; Standing over night, centrifugal or filtration, the collecting precipitation thing, dehydrated alcohol, acetone replace washed twice, and 60 ℃ of vacuum-dryings promptly get refining astragalus polysaccharide 1.8g, and yield is 9 ‰.
Get the refining ultrasonic water dissolution of polysaccharide of the 0.5g Radix Astragali, carry out DEAE Sepharose Fast Flow weak anionic exchange column chromatography, deionized water wash-out, flow velocity are 1.0mLmin
-1The phenol sulfuric acid process detects, and merging the 490nm place has absorption portion, concentrates, and lyophilize obtains white powder astragalus polysaccharides 90mg.With the water-soluble after Sephacryl S-200 gel filtration chromatography purifies and separates of astragalus polysaccharides, deionized water wash-out, flow velocity are 9mLh
-1, collect water elution liquid, the HPLC-ELSD monitoring merges the longest single symmetrical peak part of retention time, concentrates, and lyophilize promptly gets white astragalus polysaccharides 37mg, and yield is 0.67 ‰.
The gained astragalus polysaccharides is measured through HPLC-ELSD HPGPC method, and molecular-weight average is 9000; 5mL 1molL
-1H
2SO
4Behind 100 ℃ of following water-bath tube sealing hydrolysis 6h, the method inspection with ply of paper chromatography and standard substance contrast only contains D-glucose in the hydrolyzed solution, shows that this astragalus polysaccharides only is made up of glucose, is homopolysaccharide.
Embodiment 3
Get Milkvetch Root meal 500g, add 1500mL 95% ethanol (v/v) backflow degreasing 1.5h, filter, add 6000mL water refluxing extraction in the residue three times, each 90min.The extracting solution filtered while hot, merging filtrate is evaporated to 1800mL.Add 95% ethanol (v/v) under 4 ℃ in concentrated solution, stir, the regulator solution determining alcohol is 80%, standing over night.Centrifugal, collecting precipitation, dehydrated alcohol, acetone replace washed twice, and 60 ℃ of vacuum-dryings get pale brown toner powder Radix Astragali Crude polysaccharides 75g, and yield is 15%.
Get Crude polysaccharides 20g and be mixed with 20gL
-1Aqueous solution 1000mL adds stomach en-100g, and 37 ℃ of following enzymolysis 6h boil 10min and make unnecessary proteolytic enzyme sex change settle out centrifugal removing.Adding Sevage reagent in resulting supernatant solution (chloroform: the volume ratio of propyl carbinol=4: 1) 150mL, the violent jolting 20～30min of mixture, centrifugal, collect the upper strata aqueous phase solution.Repeat this process 6 times.Adding 15g DEAE Mierocrystalline cellulose to concentration expressed in percentage by weight in the aqueous phase solution of upper strata is 1.5%, boils 10min, removes by filter Mierocrystalline cellulose, and filtrate is concentrated into about 100mL.Concentrated solution is that 3000 dialysis tubing is dialysed with the molecular weight that dams, altogether 3d.Add 95% (v/v) ethanolic soln in the solution after dialysis, the regulator solution determining alcohol is that 80% (v/v) precipitates; Standing over night, centrifugal or filtration, the collecting precipitation thing, dehydrated alcohol, acetone replace washed twice, and 60 ℃ of vacuum-dryings promptly get the refining polysaccharide 1.5g of the Radix Astragali, and refined yield is 11 ‰.
Get the ultrasonic water-soluble back of the refining polysaccharide of the 0.5g Radix Astragali by DEAE Sepharose Fast Flow weak anionic exchange column chromatography, use the deionized water wash-out, flow velocity is 1.2mLmin
-1, wash-out 8h.The phenol sulfuric acid process detects, and merging the 490nm place has absorption portion, concentrates, and lyophilize obtains white powder astragalus polysaccharides 100mg.Astragalus polysaccharides is water-soluble after Sephacryl S-200 gel filtration chromatography separates, and deionized water wash-out, flow velocity are 10mLh
-1, collect water elution liquid, the HPLC-ELSD monitoring merges the longest single symmetrical peak part of retention time, concentrates, and lyophilize promptly gets white astragalus polysaccharides 40mg, and yield is 0.9 ‰.
The gained astragalus polysaccharides is measured through HPLC-ELSD HPGPC method, and molecular-weight average is 9000; 5mL 1molL
-1H
2SO
4Behind 100 ℃ of following water-bath tube sealing hydrolysis 6h, the method inspection with ply of paper chromatography and standard substance contrast only contains D-glucose in the hydrolyzed solution, shows that this astragalus polysaccharides only is made up of glucose, is homopolysaccharide.
Embodiment 4
This case step is with embodiment 3,, difference is that DEAE weak anionic exchange column filler wherein is DEAE Cellulose, the gel chromatography filler is Sephacryl S-200.Final white radix astragali homopolysaccharide 37mg, the yield 0.84 ‰ of getting.
Embodiment 5
This case step is with embodiment 3,, difference is that DEAE weak anionic exchange column filler wherein is DEAE Sepharose Fast Flow, the gel chromatography filler is Superose 12PG, final white radix astragali homopolysaccharide 41mg, yield 0.91 ‰.
Embodiment 6
This case step is with embodiment 3,, difference is that DEAE weak anionic exchange column filler wherein is DEAE Sephadex A-50, the gel chromatography filler is Sephadex G-150, final white radix astragali homopolysaccharide 42mg, yield 0.95 ‰.
Claims (4)
1. the preparation method of a radix astragali homopolysaccharide is characterized in that, comprises the steps:
Step 1 is got radix astragali coarse powder, and with the alcohol reflux degreasing 1.5h of 3 times of amount volume parts, wherein ethanol is that volume fraction is 95% ethanol; Filter, be incorporated as the water refluxing extraction 90min of 6～12 times of amounts of dregs of a decoction volume parts in the dregs of a decoction, filter, repeat to extract merging filtrate 2 times;
Step 2 is got filtrate, is concentrated into 1/5～1/10 of filtrate volume, obtain concentrated solution, the adding volume fraction is 95% ethanol in concentrated solution, stirs, alcoholic acid volume fraction in solution is 50%～80%, precipitation, standing over night, centrifugal or filtration, the collecting precipitation thing, washing, 60 ℃ of vacuum-dryings promptly get Radix Astragali Crude polysaccharides;
Step 3, the Radix Astragali Crude polysaccharides that utilizes step 2 to obtain prepares 20gL
-1The Radix Astragali Crude polysaccharides aqueous solution, be that 1: 1～1: 5 ratio adds stomach en-according to Radix Astragali Crude polysaccharides and pepsic mol ratio in the Radix Astragali Crude polysaccharides aqueous solution, 37 ℃ of following enzymolysis 3～7 hours boil 10min, and are centrifugal, obtain supernatant liquor;
Step 4 is 4: 1～6: 1 a ratio in supernatant liquor and Sevage reagent volume ratio, adds Sevage reagent in supernatant liquor, and wherein Sevage reagent is specially: chloroform and propyl carbinol be by volume 4: 1 formulated; Shaking out, centrifugal and collection upper strata aqueous phase solution, repetitive operation 3～6 times;
Step 5, in the aqueous phase solution that step 4 obtains, add diethylaminoethyl cellulose, making the weight percent of diethyllaminoethyl in aqueous phase solution is 0.5%～1.5%, boil 10～20min, filter, obtain filtrate, filtrate is concentrated into 1/10 of Radix Astragali Crude polysaccharides aqueous solution volume in the step 3, concentrated solution is that 3000 dialysis tubing is dialysed with the molecular weight that dams, and dialysis 3d adds volume fraction and be 95% ethanol in the solution after dialysis, the alcoholic acid volume fraction is 80% in solution, precipitation, standing over night, centrifugal or filtration, the collecting precipitation thing, washing, 60 ℃ of vacuum-dryings promptly get the refining astragalus polysaccharide;
Step 6 is got the refining astragalus polysaccharide, ultrasonic water dissolution, and the weak negative ion-exchange chromatography of diethyllaminoethyl separates, and deionized water wash-out, flow velocity are 0.8～1.2mLmin
-1, the phenol sulfuric acid process detects, and merges the part that there is absorption at the 490nm place, concentrates, and lyophilize obtains the white solid thing;
Step 7 is dissolved in water the white solid thing that obtains in the step 6, and through the gel filtration chromatography purifies and separates, deionized water wash-out, flow velocity are 8～10mLh
-1, the HPLC-ELSD monitoring merges the longest symmetrical peak part of retention time according to the liquid phase collection of illustrative plates, concentrates, and lyophilize promptly gets radix astragali homopolysaccharide.
2. the preparation method of radix astragali homopolysaccharide according to claim 1 is characterized in that, in step 2 and the step 5, described washing is that elder generation washes once with dehydrated alcohol, washes once with acetone again, repeats twice.
3. the preparation method of radix astragali homopolysaccharide according to claim 1 is characterized in that, in the step 3, and described stomach en-enzyme activity 〉=1200U/g.
4. the preparation method of radix astragali homopolysaccharide according to claim 1 is characterized in that, the water of described column chromatography is a deionized water.
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| CN109265576A (en) * | 2018-09-27 | 2019-01-25 | 广东药科大学 | A kind of macromolecule astragalus polyose and the preparation method and application thereof with anti-inflammatory activity |
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