CN102653568A - Herba taraxaci homogeneous polysaccharide and preparation method and application thereof - Google Patents
Herba taraxaci homogeneous polysaccharide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses herba taraxaci homogeneous polysaccharide and a preparation method and application thereof. The herba taraxaci homogeneous polysaccharide is prepared by the following steps: performing water extraction and alcohol precipitation and dialysis on herba taraxaci, capturing components of which the molecular weights are more than 10,000 Daltons; separating through anion exchange column chromatography; and purifying through a sephadex chromatographic column. The molecular weight of the homogeneous polysaccharide is 1.6*10<4> Daltons; and monosaccharide mainly comprises arabinose, glucose, galactose, little xylose, rhamnose and mannose. A pharmacodynamics experiment shows that the CP50 value of the classical pathway anticomplement activity is 0.0126 mg/mL, the AP50 value of the alternative pathway anticomplement activity is 0.05885 mg/mL, and the effective removal rate of DPPH free radical is up to 63 percent; and the herba taraxaci homogeneous polysaccharide is expected to be used as an anticomplement active component or/and an antioxidant active component for preparing a health-protetcion food or a medicament preparation and has a wide application prospect and high market value.
Description
Technical field
The present invention relates to a kind of taraxacum homogeneous polysaccharide, belong to technical field of traditional Chinese medicines.
Background technology
Taraxacum (Herba Taraxaci) is the composite family perennial herb, another name Herba crotalariae albidae, Rhizoma Trillii Tschonoskii.Have clearing heat and detoxicating, dispersing swelling and dissipating binds, inducing diuresis for treating stranguria syndrome is used for furuncle swelling toxin, acute mastitis, scrofula, hot eyes, pharyngalgia, lung carbuncle, acute appendicitis, jaundice due to damp-heat, heat is drenched puckery pain.Modern pharmacological research shows that taraxacum has cholagogic and protects the liver, and antiendotoxin is anticancer, and is hypoglycemic, anticoagulation, diuresis, strengthening the spleen and stomach, prebiotics, effects such as broad-spectrum antibacterial and immunological enhancement.
Taraxacum is studied existing history for a long time at home and abroad as medicinal plant, and taraxacum has abundant nutritive value, contains protein, fat, glucide and VITAMINs etc.(Xu Dan etc., the chemical research of taraxacum, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2004,29 (3): 229-230).Up to now; Reported taraxacum chemical ingredients mainly contains (Yuan Jin etc. such as flavonoid, triterpenes, phytosterol, coumarins, the long-chain fat same clan, sesquiterpene lactones class, pigment, volatile oil, choline, acetonyl ester class, phenolic acids, chlorogenicacid, coffic acid, organic acid, amino acid and mineral substance; Wild plant taraxacum Study on nutrition; Amino acid and Biological resources; 2006,28 (2): 22-23), concrete as: taraxasterol (Taraxasterol), choline (Choline), synanthrin (Inulin), pectin (Pectin); Taraxol (Taraxol), Stigmasterol (Stigmasterol), β-amyrin (β-Amyrin), β-Gu Zaichun (β-Sitosterol), taraxerol (taraxerol), taraxacin (Taraxacerin), taraxacin (Taraxacin) and vitamin A, B, C etc.
Complement system is one of immune defense system of wanting of body weight for humans.The normal activation of complement system is being eliminated external mikrobe, is being removed damage in the human body or dead cell and tissue and keep in the physiological processs such as balance of body and play an important role.Yet the improper activation of this system can cause human immune system's overreaction, causes the damage and the inflammatory reaction of human body self healthy tissues, is the important medium of inflammatory reaction.Research shows that the disease relevant with the complement excessive activation relates to multiple diseases such as rheumatoid arthritis, senile dementia, adult respiratory distress syndrome and systemic lupus erythematous.Report is arranged; Severe atypical pneumonia (SARS) and the bird flu that is caused by the infection of H5N1 C-type virus C can be found immune overreaction symptom, clinically like ARDS; Septic shock and Reyes syndrome etc., above-mentioned reaction symptom is relevant by people and cellular immunization, humoral immunization excessive activation.In view of the vital role of complement excessive activation in causing human many critical illness, therefore, treatment has crucial clinical meaning to complement system activated inhibition.
Extensively there is activeconstituents in the natural drug with anticomplementary action; Accomplished the screening of part natural drug such as Chinese ephedra, genseng, the bark of eucommia etc. both at home and abroad; Finding that the chemicals such as some polysaccharide, protein, polypeptide, steroid class, terpene and vegeto-alkali that occurring in nature extensively exists have significant ACA, is many with glycosylated protein and saccharan compound wherein.Heparin is a sulfated polysaccharides that has uronic acid and GS to be polymerized, and is to study morning, more sophisticated complement inhibitor.But because heparin has blood coagulation resisting function, need higher concentration competence exertion drug effect in vivo, and spinoff is big, has limited its clinical application.Application number is that 200710046222.7 Chinese patent document discloses a kind of vegetable polysaccharides with ACA; Adopt the mixed polysaccharide of five kinds of polysaccharide; By Herba Houttuyniae, Wild Chrysanthemum, oriental wormwood, eupatorium and tsaoko preparation; Act on C1Q, C1r, C1s, C2, C3, C4, C5, C9 component, and do not have blood coagulation resisting function.Application number is that 200710044099.5 Chinese patent document discloses the purposes of a kind of wooden element that extracts from the bark of eucommia at the preparation anticomplement medicament.Application number is that 200910201362.6 Chinese patent document discloses the purposes of a kind of flavonoid that extracts from kuh-seng at the preparation anticomplement medicament, and the flavonoid compound of gained all has stronger restraining effect to the classical pathway and the alternative pathway of complement system.But do not see the correlative study report of the ACA of taraxacum and polysaccharide thereof so far.
Summary of the invention
To the problems referred to above that prior art exists, the purpose of this invention is to provide a kind of taraxacum homogeneous polysaccharide, to widen the application of taraxacum polysaccharide.
For realizing the foregoing invention purpose, the technical scheme that the present invention adopts is following:
A kind of taraxacum homogeneous polysaccharide, by the taraxacum herb through water extract-alcohol precipitation, dialysis intercepting molecular weight greater than 10000 daltonian components, separate through anion exchange chromatography then, get through the sephadex chromatography column purification again; The molecular weight of said homogeneous polysaccharide is 1.6 * 10
4Dalton, its monose consists of: the D-pectinose accounts for 25.4wt%, D-glucose and accounts for 17.7wt%, D-semi-lactosi and account for 44.5wt%, D-wood sugar and account for that 4.1wt%, D-rhamnosyl account for 3wt%, the D-seminose accounts for 5.3wt%.
A kind of preparation method of said taraxacum homogeneous polysaccharide comprises the steps:
A) at first the taraxacum herb is used the alcohol reflux degreasing, filter then, volatilize the ethanol in the dregs of a decoction; Soak, decoct extraction; United extraction liquid concentrates, and is centrifugal, collects supernatant; Add ethanol and carry out precipitating; Centrifugal must the deposition, and volatilize ethanol wherein; The water dissolution precipitation, it is that 10000 daltonian dialysis tubings were dialysed in water 24～72 hours that gained solution uses molecular weight cut-off; Concentrate dialysate, lyophilize gets taraxacum polyoses extract bullion;
B) above-mentioned taraxacum polyoses extract bullion is dissolved in water, centrifugal; To precipitate and add the water heating for dissolving again, centrifugal; Merge two times centrifugal gained supernatant, be splined on diethylin ethyl (DEAE) anion-exchange column, use the NaCl eluant solution of zero(ppm) water, 0.2,0.5,0.8,1.0mol/L successively, collect elutriant by the NaCl eluant solution of 1.0mol/L; Concentrating under reduced pressure, using molecular weight cut-off then is that 3500 daltonian dialysis tubings carry out flowing water dialysis 24 hours; Being splined on gel chromatography column, is elutriant with the NaCl solution of 0.2mol/L, flow velocity 1.1mL/min; Differential refraction detects, and protein purification system is collected homogeneous polysaccharide at line imaging according to the gel separation collection of illustrative plates; Using molecular weight cut-off again is that 3500 daltonian dialysis tubings carry out deionized water dialysis 48 hours; Concentrate dialysate, lyophilize promptly gets described taraxacum homogeneous polysaccharide.
As further preferred version, the operation of said backflow degreasing is following: the adding volumn concentration is 95% ethanol in the taraxacum herb, and the ethanol volume of adding is 2～4 times of taraxacum herb quality; Refluxed then 1～3 hour.
As further preferred version, the operation of said extraction is following: in the dregs of a decoction, add deionized water, in soaking at room temperature 1～3 hour, heated and boiled decocted 2～4 hours then, filtered, and the gained dregs of a decoction are repeated aforesaid operations 1～3 time again; Add volume of water at every turn and be 8～10 times of dregs of a decoction quality.
As further preferred version, the specific density that extracting solution is concentrated at 60～80 ℃ is 1.0～1.5.
As further preferred version, the operation of said precipitating is following: in supernatant, to add volumn concentration down be 95% ethanol constantly stirring, and alcoholic acid adding volume is 2～4 times of supernatant volume; Finish, left standstill 12～24 hours at 0～5 ℃.
A kind of purposes of taraxacum homogeneous polysaccharide of the present invention is to be used to prepare protective foods or pharmaceutical prepn with said taraxacum homogeneous polysaccharide as the ACA composition.
The another kind of purposes of taraxacum polyoses extract of the present invention is to be used to prepare protective foods or pharmaceutical prepn with said taraxacum homogeneous polysaccharide as oxidation-resistant active ingredient.
Described pharmaceutical prepn can be any formulation that is suitable for clinical use, comprises solid preparation (like capsule, tablet, granule etc.), semi-solid preparation (like ointment etc.), liquid preparation (like oral liquid, suspensoid, emulsion etc.), injection etc.
Compared with prior art, the present invention has following beneficial effect:
Pharmacodynamic experiment shows: the CP of taraxacum homogeneous polysaccharide classical pathway ACA provided by the present invention
50Value is 0.0126mg/mL, and 0.0206mg/mL compares with the positive drug heparin, shows extraordinary ACA; The AP of alternative pathway ACA
50Value is 0.05885mg/mL, compares with positive drug heparin 0.0887mg/mL and also shows extraordinary ACA; Its ACA target spot is C1q, C1r, C1s, C2, C3, C4 and C5; In addition, taraxacum homogeneous polysaccharide provided by the present invention to the effective clearance rate of DPPH radical up to 63%.Therefore; Taraxacum homogeneous polysaccharide of the present invention is expected as the ACA composition or/and oxidation-resistant active ingredient is used to prepare protective foods or pharmaceutical prepn, and is safe in utilization, the sorrow that has no side effect; And preparation technology is simple; Steady quality is suitable for large-scale production, has broad application prospects and marketable value.
Description of drawings
Fig. 1 is the gel separation figure of the taraxacum homogeneous polysaccharide described in the present invention;
The HPLC-GPC liquid phase collection of illustrative plates of the taraxacum homogeneous polysaccharide that Fig. 2 makes for the embodiment of the invention 1;
The GC-MS figure of the taraxacum homogeneous polysaccharide that Fig. 3 makes for the embodiment of the invention 1;
GC-MS figure after the taraxacum homogeneous polysaccharide that Fig. 4 makes for the embodiment of the invention 1 methylates;
The classical pathway hemolytic activity graphic representation of the taraxacum homogeneous polysaccharide that Fig. 5 makes for the embodiment of the invention 1;
The alternative pathway hemolytic activity graphic representation of the taraxacum homogeneous polysaccharide that Fig. 6 makes for the embodiment of the invention 1;
The ACA target spot analytical results figure of the taraxacum homogeneous polysaccharide that Fig. 7 makes for the embodiment of the invention 1.
Embodiment
Below in conjunction with embodiment to the present invention do further in detail, intactly explanation.
Embodiment 1
Get the taraxacum herb and place in the extractor, the adding volumn concentration is 95% ethanol, and the ethanol volume of adding is 2 times of taraxacum herb quality; The degreasing 2 hours that refluxes is filtered, and volatilizes the ethanol in the dregs of a decoction; In the dregs of a decoction, add deionized water, in soaking at room temperature 3 hours, heated and boiled decocted 4 hours then, filtered, and the gained dregs of a decoction are repeated aforesaid operations 3 times again; Add volume of water at every turn and be 10 times of dregs of a decoction quality; United extraction liquid, the specific density that extracting solution is concentrated at 80 ℃ is 1.2, and is centrifugal, collects supernatant; In supernatant, to add volumn concentration down be 95% ethanol constantly stirring, and alcoholic acid adding volume is 4 times of supernatant volume; Finish, insert 4 ℃ of refrigerators and left standstill 12 hours; Centrifugal must the deposition, and volatilize ethanol wherein; The water dissolution precipitation, it is that 10000 daltonian dialysis tubings were dialysed in water 72 hours that gained solution uses molecular weight cut-off, to remove micromolecular polysaccharide, albumen, ethanol and salt and some water-soluble impurities wherein; Concentrate dialysate, lyophilize gets taraxacum polyoses extract bullion, and yield is about 7.13% of herb quality.
Above-mentioned taraxacum polyoses extract bullion is dissolved in water, in the centrifugal 10min of 8500rpm; To precipitate and add the water heating for dissolving again, once more in the centrifugal 10min of 8500rpm; Merge two times centrifugal gained supernatant, be splined on diethylin ethyl (DEAE) anion-exchange column, use the NaCl eluant solution of zero(ppm) water, 0.2,0.5,0.8,1.0mol/L successively, collect elutriant by the NaCl eluant solution of 1.0mol/L; Concentrating under reduced pressure, using molecular weight cut-off then is that 3500 daltonian dialysis tubings carry out flowing water dialysis 24 hours; (60cm, 26mm) separation and purification are elutriant with the NaCl solution of 0.2mol/L further to use gel chromatography column superdex-200; Flow velocity 1.1mL/min, the shodex differential refraction detects, and protein purification system Versatiler system is at line imaging; Collect homogeneous polysaccharide according to gel separation collection of illustrative plates (seeing shown in Figure 1); Using molecular weight cut-off is that 3500 daltonian dialysis tubings carry out deionized water dialysis 48 hours, concentrate dialysate, lyophilize; Promptly get described taraxacum homogeneous polysaccharide, yield is about 5.32% of taraxacum polyoses extract bullion quality.
One, carries out purity and molecular weight detection
Detect with efficient gel permeation chromatography (HPGPC), moving phase is pure water or 0.2mol/L NaCl solution, and flow velocity is 0.8mL/min, and column temperature is 40 ℃, and detector is the differential detector, and chromatographic column is Shodex KS-805 and KS-804 series connection.With the dextran standard production standard curve of different molecular weight, per sample elution time and typical curve contrast then is with its molecular weight of Agilent GPC computed in software.
Fig. 2 is the HPLC liquid phase collection of illustrative plates of the taraxacum homogeneous polysaccharide that obtained, is visible as single symmetrical peak (is moving phase with 0.2mol/L NaCl solution), shows that it is a homogeneous polysaccharide;
The molecular weight that Agilent GPC software records the gained homogeneous polysaccharide is 1.6 * 10
4Dalton's (is reference standard with the dextran standard).
Two, monose is formed detection
1) instrument and reagent
Thermo fisher DSQ gas chromatograph, (305m * 0.32mm * 0.5 μ m) chromatographic column.
Ethanol, acetonitrile, chloroform, diacetyl oxide, SODIUM SULPHATE ANHYDROUS 99PCT, trichoroacetic acid(TCA), trifluoroacetic acid, 4-methylmorphine borine are analytical pure.
2) reference substance
D-rhamnosyl, L-Fucose, D-wood sugar, D-pectinose, D-seminose, D-glucose, D-semi-lactosi, reference substance (sigma company).
3) preparation of reference substance solution
Carry out sugared compositional analysis with the reductive water solution: fixed each the monose reference substance 1.0mg of accurate title places long test tube; Adding freshly prepared concentration is the 4-methylmorpholine borine aqueous solution 50 μ L of 80mg/mL and the trifluoroacetic acid aqueous solution 200 μ L of 3mol/L, adds heat 5 minutes in 80 ℃ behind the mixing; Treat to add the 50 μ L 4-methylmorpholine borine aqueous solution again after the solution cooling, 120 ℃ were heated 1 hour; Treat to add the 100 μ L 4-methylmorpholine borine aqueous solution again after the solution cooling, 50 ℃ of evaporates to dryness add 3mL acetonitrile evaporate to dryness 3 times again; Add each 100 μ L of aceticanhydride and trifluoroacetic acid then, acetylize is 10 minutes in 50 ℃ of water-baths, adds zero(ppm) water 2mL and chloroform 2mL extraction, and water is given a baby a bath on the third day after its birth time again, crosses the SODIUM SULPHATE ANHYDROUS 99PCT post, carries out the GC-MS analysis.
4) preparation of need testing solution
Carry out the monose compositional analysis with the reductive water solution: accurate title is decided gained homogeneous polysaccharide 1.0mg and is placed long test tube; Adding freshly prepared concentration is the 4-methylmorpholine borine aqueous solution 50 μ L of 80mg/mL and the trifluoroacetic acid aqueous solution 200 μ L of 3mol/L, heats 5 minutes in 80 ℃ behind the mixing; Treat to add the 50 μ L 4-methylmorpholine borine aqueous solution again after the solution cooling, 120 ℃ were heated 1 hour; Treat to add the 100 μ L 4-methylmorpholine borine aqueous solution again after the solution cooling, 50 ℃ of evaporates to dryness add 3mL acetonitrile evaporate to dryness 3 times again; Add each 100 μ L of aceticanhydride and trifluoroacetic acid then, acetylize is 10 minutes in 50 ℃ of water-baths, adds zero(ppm) water 2mL and chloroform 2mL extraction, and water is given a baby a bath on the third day after its birth time again, crosses the SODIUM SULPHATE ANHYDROUS 99PCT post, carries out the GC-MS analysis.
5) GC conditions
The GC-MS analysis condition is: TR-5MS (Thermo) chromatographic column (60m * 0.25mm * 0.25 μ m); The temperature programming condition is: since 140 ℃ of column temperatures, be warming up to 198 ℃ with 2 ℃/min, insulation 4min is warming up to 214 ℃ with 4 ℃/min again, is warming up to 217 ℃ with 1 ℃/min then, keeps 4min, is warming up to 250 ℃ with 3 ℃/min at last, keeps 5min; Injector temperature is 250 ℃; Carrier gas is He, and flow velocity is 1mL/min.
6) measure
Adopt vapor-phase chromatography to measure each reference substance and a confession appearance test sample solution, the confession appearance test product collection of illustrative plates that obtains and the contrast of the collection of illustrative plates of standard monose respectively.Comparison through RT can know, the monose of said homogeneous polysaccharide is formed and is mainly D-pectinose and D-glucose, D-semi-lactosi, contains a little D-wood sugar in addition, D-rhamnosyl, D-seminose.
Fig. 3 is the GC-MS figure of described taraxacum homogeneous polysaccharide, can know through area normalization method: the D-pectinose accounts for 25.4wt%, D-glucose and accounts for 17.7wt%, D-semi-lactosi and account for 44.5wt%, D-wood sugar and account for that 4.1wt%, D-rhamnosyl account for 3wt%, the D-seminose accounts for 5.1wt%.
Three, methylation analysis
1) methylation reaction
Getting gained homogeneous polysaccharide 20mg is put in the 25mL triangular flask; Dried overnight in being placed with the moisture eliminator of Vanadium Pentoxide in FLAKES adds 2mL DMSO 99.8MIN. (DMSO) stirring and dissolving 10min, is insoluble to DMSO like sample; Can be under the 40KHz ultrasonic frequency ultrasonic 10min; The NaOH 200mg that adds grinding powder again, ultrasonic 10min (40KHz), 25 ℃ of stirring at room were reacted 1 hour; Dropwise add CH
3I 1.5mL was placed on the dark place stirring reaction 1 hour after adding; Add 2mL zero(ppm) water at last and decompose residual CH
3I, stirring reaction 10min is washed till in the 60mL separating funnel with zero(ppm) water, with chloroform extraction three times; Combining extraction liquid, it is inferior to give a baby a bath on the third day after its birth with zero(ppm) water, tells chloroform layer, and low temperature revolves dried; Dry then, repeat above-mentioned methylation reaction 3 times, obtain the homogeneous polysaccharide sample of exhaustive methylation.
2) test agent is prepared
Get the homogeneous polysaccharide sample 2mg of exhaustive methylation, dissolve in the trifluoroacetic acid aqueous solution of adding 3mL 2mol/L, change in the 10mL ampoule; Seal, hydrolysis reaction is 2 hours in 121 ℃ of baking ovens, takes out; Evaporated under reduced pressure adds the methyl alcohol evaporate to dryness three times again, and repetitive operation is to eliminate TFA fully; Resistates adds 100mg NaBH after the hydrolysis
4With 2mL water, the room temperature reduction reaction is spent the night; Add the Glacial acetic acid min. 99.5 reaction to remove excessive N aBH
4, on Rotary Evaporators, be concentrated into thick liquid, add methyl alcohol-Glacial acetic acid min. 99.5 (volume ratio 5:1) 3～5mL; Evaporate to dryness three times adds methyl alcohol evaporate to dryness twice again, gets white powder; Put into 105 ℃ of 15min of baking oven to remove moisture, add the 3mL diacetyl oxide, acetylization reaction is 1 hour in 101 ℃ of baking ovens; Take out, repeatedly add 50 ℃ of water-bath decompressions of toluene azeotropic evaporate to dryness to Powdered; Add the suitable quantity of water dissolving, add chloroform extraction three times, the combined chloroform layer with water washing three times, is told chloroform layer, carries out the GC-MS analysis with anhydrous sodium sulfate drying, after concentrated.
3) chromatographic condition
The GC-MS analysis condition is: TR-5MS (Thermo) chromatographic column (60m * 0.25mm * 0.25 μ m); The temperature programming condition is: since 140 ℃ of column temperatures, be warming up to 180 ℃ with 2 ℃/min, be warming up to 200 ℃ with 1 ℃/min again, be warming up to 250 ℃ with 3 ℃/min at last, keep 5min; Injector temperature is 250 ℃; Carrier gas is He, and flow velocity is 1mL/min.Concrete GC-MS collection of illustrative plates is seen shown in Figure 4.
4) analytical results
Concrete analysis is the result see shown in the table 1.
The methylation analysis result of the said homogeneous polysaccharide of table 1
| Mode of connection | Methylated saccharide residue | Mol ratio | tR/min |
| 1- |
2,3,4-Me3-Rhap | 5.21 | 21.5 |
| 1- |
2,3,5-Me3-Araf | 2.04 | 18.51 |
| 1，5- |
2,3-Me2-Araf | 3.41 | 19.6 |
| 1- |
2,3,4,-Me3-Ara | 5.19 | 20.14 |
| 1- |
2,3,4,6-Me4-Manp | 4.72 | 24.8 |
| 1,6- |
2,3,4-Me3-Manp | 4.63 | 26.5 |
| 1- |
2,3,4,6-Me4-Glcp | 8.06 | 26.97 |
| 1,4- |
2,3,6-Me3-Glcp | 5.97 | 27.4 |
| 1- |
2,3,4,6-Me4-Galp | 6.46 | 28.22 |
| 1,4- |
2,3,6-Me3-Galp | 22.14 | 33.09 |
Visible by table 1: D-rhamnosyl (Rha) exists end group to be connected with D-wood sugar (Xyl); And D-pectinose (Ara) is with 1-Araf and α-1, two kinds of mode of connection of 5-Araf; D-seminose (Man) exists end group connection and 1, and 6-connects two kinds of mode of connection; D-glucose (Glc) exists end group to connect and β-1, and 4-connects two kinds of mode of connection; The topmost mode of connection of semi-lactosi (Gal) is β-1, and 4-Galp is connected with T-Galp.The main chain that described homogeneous polysaccharide is described mainly is to be made up of the D-semi-lactosi that β (1 → 4) connects, the D-glucose that connects of some β (1 → 4) in addition, the D-pectinose that is connected with α (1 → 5).
Four, carrying out the anticomplement pharmacodynamics detects
1) the hemolytic activity measuring method of classical pathway of complement
Get the complement and the trial-product mixing of definite threshold concentration, add an amount of BBS (barbitol buffer solution), hemolysin and 2%SRBC (sheep red blood cell (SRBC)), polysaccharide control group, complement group and full haemolysis group are set simultaneously, concrete proportioning is according to shown in the table 2.
Behind 37 ℃ of water-bath 30min, get every pipe supernatant 0.2mL respectively behind 5000r/min, 4 ℃ of following centrifugal 10min, under 405nm, measure absorbancy with ELIASA.
To detect substrate concentration as the X axle, the haemolysis inhibiting rate calculates CP as the mapping of Y axle
50Value.
Table 2 complement classics add the volume (mL) of each composition in hemolytic experiment
| Divide into groups | BBS | Tri-distilled water | Complement | | Hemolysin | 2%SRBC | |
| The polysaccharide determination group | 0.2 | - | 0.1 | ?0.1 | 0.1 | 0.1 | |
| The polysaccharide control group | 0.5 | - | ?- | ?0.1 | ?- | ?- | |
| The complement group | 0.3 | - | 0.1 | - | 0.1 | 0.1 | |
| Full haemolysis group | ?- | ?0.5 | ?- | - | ?- | ?- |
Detected result is seen shown in Figure 5, is shown by Fig. 5: the CP of the classical pathway of complement of positive drug heparin
50Be 0.0206mg/mL, the CP of the classical pathway of complement of said homogeneous polysaccharide
50Be 0.0126mg/mL; The CP of taraxacum homogeneous polysaccharide of the present invention
50Value shows further that much smaller than the positive drug heparin its ACA is superior to the positive drug heparin, can effectively suppress the haemolysis that the complement excessive activation causes.
2) the hemolytic activity measuring method of alternative pathway of complement
Get the complement and the trial-product mixing of definite threshold concentration, add an amount of 0.5%RE (rabbit erythrocyte), polysaccharide control group, complement group and full haemolysis group are set simultaneously, concrete proportioning is seen shown in the table 3.
Behind 37 ℃ of water-bath 30min, get every pipe supernatant 0.2mL respectively behind 5000r/min, 4 ℃ of following centrifugal 10min, under 405nm, measure absorbancy with ELIASA.To detect substrate concentration as the X axle, the haemolysis inhibiting rate calculates AP as the mapping of Y axle
50Value.
Table 3 alternative complement pathway by way of each assembly of hemolytic experiment than (unit: mL)
| Divide into groups | The AP diluent | Tri-distilled water | Complement | Homogeneous polysaccharide | 0.5%RE |
| The polysaccharide determination group | ?- | - | 0.15 | 0.15 | 0.2 |
| The polysaccharide control group | 0.35 | - | - | 0.15 | ?- |
| The complement group | 0.15 | - | 0.15 | ?- | 0.2 |
| Full haemolysis group | ?- | ?0.3 | - | ?- | 0.2 |
Detected result is seen shown in Figure 6, is shown by Fig. 6: 50% inhibition concentration AP of said homogeneous polysaccharide
50Be 0.05885mg/mL, the AP of positive drug heparin
50Be 0.0887mg/mL, show that further the alternative pathway ACA of taraxacum homogeneous polysaccharide of the present invention is superior to the positive drug heparin, show good ACA.
In sum, classical pathway and alternative pathway hemolytic activity detected result show that all taraxacum homogeneous polysaccharide of the present invention has comparatively ideal ACA, is superior to the positive control drug heparin.
3) action target spot of complement inhibition is identified
The complement of critical weaker concn and polysaccharide trial-product mixing are provided with polysaccharide control group, disappearance serologic group and complement group, full haemolysis group simultaneously, according to adding an amount of anticomplement antiserum(antisera), 1:1000 hemolysin and 2%SRBC (sheep red blood cell (SRBC)) shown in the table 4.
With 5000rpm behind 37 ℃ of water-bath 30min of every pipe, get every pipe supernatant 0.2mL behind the centrifugal 10min respectively in 96 orifice plates, under ELIASA 405nm, measure absorbancy.Calculate hemolysis rate after deducting corresponding polysaccharide control group absorbance.Relatively lack serologic group and in corresponding disappearance serum, added the variation of the target spot test set hemolysis rate of the human serum that trial-product handled; According to target spot test set haemolysis situation, judge that trial-product has or not antagonistic action to C1Q, C1r, C1s, C2, C3, C4, C5 and C9.The target spot test set lacks the serologic group haematolysis ability more accordingly and has recovered, and explains that trial-product does not act on this disappearance component; If haematolysis ability can not recover, explain that then trial-product acts on this disappearance component.
Each assembly of action target spot identification experiment that table 4 complement suppresses is than (unit: mL)
| Experiment is divided into groups | The target spot test set | The polysaccharide group | The polysaccharide control group | The disappearance serologic group | The complement group | Full haemolysis group |
| BBS | ?- | 0.2 | 0.5 | 0.2 | 0.3 | 0.5 |
| Tri-distilled water | ?- | ?- | ?- | ?- | ?- | ?- |
| Complement | 0.1 | 0.1 | ?- | ?- | 0.1 | ?- |
| Polysaccharide | 0.1 | 0.1 | 0.1 | ?- | ?- | ?- |
| Hemolysin | 0.1 | 0.1 | ?- | 0.1 | 0.1 | ?- |
| Disappearance serum | 0.2 | ?- | ?- | 0.2 | ?- | ?- |
| 2%SRBC | 0.1 | 0.1 | ?- | 0.1 | 0.1 | 0.1 |
Detected result is seen shown in Figure 7; Shown by Fig. 7: adding target spot test set C1q, C1r, C1s, C2, C3, C4 and C5 lack the serologic group haematolysis ability more accordingly and can not recover; Showing has antagonistic action, explains that the polysaccharide trial-product acts on C1q, C1r, C1s, C2, C3, C4 and C5; After adding target spot test set C9, lack the serologic group haematolysis ability more accordingly and recover, explain that trial-product does not act on this disappearance group C9; Thereby the ACA action target spot that further shows taraxacum homogeneous polysaccharide of the present invention is C1q, C1r, C1s, C2, C3, C4 and C5.
Five, carry out anti-oxidant drug effect experiment
Through the clearance rate of homogeneous polysaccharide to DPPH (1,1-phenylbenzene-2-trinitrophenyl-hydrazine) radical, detect its anti-oxidant activity, adopt vitamins C (Vc) as positive control drug, sample and reference substance concentration thereof are 500 μ g/mL.
DPPH free radical scavenging activity calculation formula is: DPPH free radical scavenging activity=A0-(A-Ab)/A0 * 100%; Wherein, each proportioning is seen shown in the table 5.
Each assembly ratio of the anti-oxidant drug effect experiment of table 5
| Experiment is divided into groups | Supply the test agent proportioning |
| A0 n.s. solution | 1mL water+2mL DPPH+2mL methyl alcohol |
| A DPPH+ sample | 1mL polysaccharide sample+2mL DPPH+2mL methyl alcohol |
| The Ab sample does not have DPPH | 1mL polysaccharide sample+4mL methyl alcohol |
Detected result shows: the DPPH free radical scavenging activity of blank sample is 1.3%, and the DPPH free radical scavenging activity of homogeneous polysaccharide is 63%, and the DPPH free radical scavenging activity of Vc is 80%; Show that further taraxacum homogeneous polysaccharide of the present invention compares with positive drug VC, anti-oxidant activity is stronger.
Get the taraxacum herb and place in the extractor, the adding volumn concentration is 95% ethanol, and the ethanol volume of adding is 3 times of taraxacum herb quality; The degreasing 2 hours that refluxes is filtered, and volatilizes the ethanol in the dregs of a decoction; In the dregs of a decoction, add deionized water, in soaking at room temperature 2 hours, heated and boiled decocted 3 hours then, filtered, and the gained dregs of a decoction are repeated aforesaid operations 2 times again; Add volume of water at every turn and be 9 times of dregs of a decoction quality; United extraction liquid, the specific density that extracting solution is concentrated at 70 ℃ is 1.1, and is centrifugal, collects supernatant; In supernatant, to add volumn concentration down be 95% ethanol constantly stirring, and alcoholic acid adding volume is 3 times of supernatant volume; Finish, insert 4 ℃ of refrigerators and left standstill 12 hours; Centrifugal must the deposition, and volatilize ethanol wherein; The water dissolution precipitation, it is that 10000 daltonian dialysis tubings were dialysed in water 72 hours that gained solution uses molecular weight cut-off, to remove micromolecular polysaccharide, albumen, ethanol and salt and some water-soluble impurities wherein; Concentrate dialysate, lyophilize gets taraxacum polyoses extract bullion, and yield is about 6.54% of herb quality.
Above-mentioned taraxacum polyoses extract bullion is dissolved in water, in the centrifugal 10min of 8500rpm; To precipitate and add the water heating for dissolving again, once more in the centrifugal 10min of 8500rpm; Merge two times centrifugal gained supernatant, be splined on diethylin ethyl (DEAE) anion-exchange column, use the NaCl eluant solution of zero(ppm) water, 0.2,0.5,0.8,1.0mol/L successively, collect elutriant by the NaCl eluant solution of 1.0mol/L; Concentrating under reduced pressure, using molecular weight cut-off then is that 3500 daltonian dialysis tubings carry out flowing water dialysis 24 hours; Concentrate dialysate is further used gel chromatography column superdex-200 (60cm, 26mm) purifying; NaCl solution with 0.2mol/L is elutriant, flow velocity 1.1mL/min, and the shodex differential refraction detects; Protein purification system Versatiler system collects homogeneous polysaccharide at line imaging according to gel separation collection of illustrative plates (seeing shown in Figure 1), and using molecular weight cut-off is that 3500 daltonian dialysis tubings carry out deionized water dialysis 48 hours; Concentrate dialysate; Lyophilize promptly gets described taraxacum homogeneous polysaccharide, and yield is about 4.5% of taraxacum polyoses extract bullion quality.
About the structural analysis content of the homogeneous polysaccharide that present embodiment obtained and ACA experiment and anti-oxidant activity experiment content all with described in the embodiment 1.
Get the taraxacum herb and place in the extractor, the adding volumn concentration is 95% ethanol, and the ethanol volume of adding is 4 times of taraxacum herb quality; The degreasing 2 hours that refluxes is filtered, and volatilizes the ethanol in the dregs of a decoction; In the dregs of a decoction, add deionized water, in soaking at room temperature 1 hour, heated and boiled decocted 2 hours then, filtered, and the gained dregs of a decoction are repeated aforesaid operations 1 time again; Add volume of water and be 8 times of dregs of a decoction quality; United extraction liquid, the specific density that extracting solution is concentrated at 60 ℃ is 1.5, and is centrifugal, collects supernatant; In supernatant, to add volumn concentration down be 95% ethanol constantly stirring, and alcoholic acid adding volume is 2 times of supernatant volume; Finish, insert 4 ℃ of refrigerators and left standstill 12 hours; Centrifugal must the deposition, and volatilize ethanol wherein; The water dissolution precipitation, it is that 10000 daltonian dialysis tubings were dialysed in water 72 hours that gained solution uses molecular weight cut-off, to remove micromolecular polysaccharide, albumen, ethanol and salt and some water-soluble impurities wherein; Concentrate dialysate, lyophilize gets taraxacum polyoses extract bullion, and yield is about 6.07% of herb quality.
Above-mentioned taraxacum polyoses extract bullion is dissolved in water, in the centrifugal 10min of 8500rpm; To precipitate and add the water heating for dissolving again, once more in the centrifugal 10min of 8500rpm; Merge two times centrifugal gained supernatant, be splined on diethylin ethyl (DEAE) anion-exchange column, use the NaCl eluant solution of zero(ppm) water, 0.2,0.5,0.8,1.0mol/L successively, collect elutriant by the NaCl eluant solution of 1.0mol/L; Concentrating under reduced pressure, using molecular weight cut-off then is that 3500 daltonian dialysis tubings carry out flowing water dialysis 24 hours; Concentrate dialysate is further used gel chromatography column superdex-200 (60cm, 26mm) separation and purification; NaCl solution with 0.2mol/L is elutriant, flow velocity 1.1mL/min, and the shodex differential refraction detects; Protein purification system Versatiler system collects homogeneous polysaccharide at line imaging according to gel separation collection of illustrative plates (seeing shown in Figure 1), and using molecular weight cut-off is that 3500 daltonian dialysis tubings carry out deionized water dialysis 48 hours; Concentrate dialysate; Lyophilize promptly gets described taraxacum homogeneous polysaccharide, and yield is about 4.1% of taraxacum polyoses extract bullion quality.
About the structural analysis content of the homogeneous polysaccharide that present embodiment obtained and ACA experiment and anti-oxidant activity experiment content all with described in the embodiment 1.
In sum, taraxacum homogeneous polysaccharide of the present invention has significant ACA and anti-oxidant activity preferably, is expected as the ACA composition or/and oxidation-resistant active ingredient is used to prepare protective foods or pharmaceutical prepn.
Be necessary at last in this explanation to be: above embodiment only is used for technical scheme of the present invention is done explanation in further detail; Can not be interpreted as the restriction to protection domain of the present invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (8)
1. taraxacum homogeneous polysaccharide is characterized in that: by the taraxacum herb through water extract-alcohol precipitation, dialysis intercepting molecular weight greater than 10000 daltonian components, separate through anion exchange chromatography then, get through the sephadex chromatography column purification again; The molecular weight of said homogeneous polysaccharide is 1.6 * 10
4Dalton, its monose consists of: the D-pectinose accounts for 25.4wt%, D-glucose and accounts for 17.7wt%, D-semi-lactosi and account for 44.5wt%, D-wood sugar and account for that 4.1wt%, D-rhamnosyl account for 3wt%, the D-seminose accounts for 5.3wt%.
2. the preparation method of the described taraxacum homogeneous polysaccharide of claim 1 is characterized in that, comprises the steps:
A) at first the taraxacum herb is used the alcohol reflux degreasing, filter then, volatilize the ethanol in the dregs of a decoction; Soak, decoct extraction; United extraction liquid concentrates, and is centrifugal, collects supernatant; Add ethanol and carry out precipitating; Centrifugal must the deposition, and volatilize ethanol wherein; The water dissolution precipitation, it is that 10000 daltonian dialysis tubings were dialysed in water 24～72 hours that gained solution uses molecular weight cut-off; Concentrate dialysate, lyophilize gets taraxacum polyoses extract bullion;
B) above-mentioned taraxacum polyoses extract bullion is dissolved in water, centrifugal; To precipitate and add the water heating for dissolving again, centrifugal; Merge two times centrifugal gained supernatant, be splined on diethylin ethyl (DEAE) anion-exchange column, use the NaCl eluant solution of zero(ppm) water, 0.2,0.5,0.8,1.0mol/L successively, collect elutriant by the NaCl eluant solution of 1.0mol/L; Concentrating under reduced pressure, using molecular weight cut-off then is that 3500 daltonian dialysis tubings carry out flowing water dialysis 24 hours; Being splined on gel chromatography column, is elutriant with the NaCl solution of 0.2mol/L, flow velocity 1.1mL/min; Differential refraction detects, and protein purification system is collected homogeneous polysaccharide at line imaging according to the gel separation collection of illustrative plates; Using molecular weight cut-off again is that 3500 daltonian dialysis tubings carry out deionized water dialysis 48 hours; Concentrate dialysate, lyophilize promptly gets described taraxacum homogeneous polysaccharide.
3. the preparation method of taraxacum homogeneous polysaccharide according to claim 2; It is characterized in that; The operation of said backflow degreasing is following: the adding volumn concentration is 95% ethanol in the taraxacum herb, and the ethanol volume of adding is 2～4 times of taraxacum herb quality; Refluxed then 1～3 hour.
4. the preparation method of taraxacum homogeneous polysaccharide according to claim 2; It is characterized in that; The operation of said extraction is following: in the dregs of a decoction, add deionized water, in soaking at room temperature 1～3 hour, heated and boiled decocted 2～4 hours then; Filter, the gained dregs of a decoction are repeated aforesaid operations 1～3 time again; Add volume of water at every turn and be 8～10 times of dregs of a decoction quality.
5. the preparation method of taraxacum polyoses extract according to claim 2 is characterized in that: the specific density that extracting solution is concentrated at 60～80 ℃ is 1.0～1.5.
6. the preparation method of taraxacum homogeneous polysaccharide according to claim 2; It is characterized in that; The operation of said precipitating is following: in supernatant, to add volumn concentration down be 95% ethanol constantly stirring, and alcoholic acid adding volume is 2～4 times of supernatant volume; Finish, left standstill 12～24 hours at 0～5 ℃.
7. the purposes of the described taraxacum homogeneous polysaccharide of claim 1 is characterized in that: be used to prepare protective foods or pharmaceutical prepn with said taraxacum homogeneous polysaccharide as the ACA composition.
8. the purposes of the described taraxacum homogeneous polysaccharide of claim 1 is characterized in that: be used to prepare protective foods or pharmaceutical prepn with said taraxacum homogeneous polysaccharide as oxidation-resistant active ingredient.
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