CN102443077B - Isostichopus badionotus fucosylated mucopolysaccharide and application thereof - Google Patents
Isostichopus badionotus fucosylated mucopolysaccharide and application thereof Download PDFInfo
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- CN102443077B CN102443077B CN201110460175.7A CN201110460175A CN102443077B CN 102443077 B CN102443077 B CN 102443077B CN 201110460175 A CN201110460175 A CN 201110460175A CN 102443077 B CN102443077 B CN 102443077B
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- Prior art keywords
- mucopolysaccharide
- fucosylation
- fucosylated
- meat ginseng
- chondroitin sulfate
- Prior art date
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- 230000000324 neuroprotective effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses an isostichopus badionotus fucosylated mucopolysaccharide of which the structural formula is shown in the specification, wherein n is equal to 70. The main chain of the isostichopus badionotus fucosylated mucopolysaccharide is in a chondroitin sulfate E structure, and the isostichopus badionotus fucosylated mucopolysaccharide has a branched chain composed of fucose sulfate; the branched chain is connected on beta-D-glucuronic acid of the main chain through a glycosidic bond; and the substitution mode of a sulfate group is mainly 2,4-O-SO4. The isostichopus badionotus fucosylated mucopolysaccharide is a chondroitin sulfate polysaccharide which is separated from sea cucumbers and has a novel structure, and can be used for preparing anti-thrombotic agents or anticoagulants.
Description
Technical field
The present invention relates to a kind of macromolecular cpd--Structural Identification and the purposes of U.S.'s meat ginseng fucosylation mucopolysaccharide, belong to natural polymer field.
Background technology
Research finds that the polysaccharide that is present in wall of sea cucumber Stichopus japonicus mainly divides two classes: the sea cucumber chondroitin sulfate (sea cucumber fucolysated chondroitin sulfate) (38 that a class is fucosylation, 39), the branch's mixed polysaccharide being formed by D-N-acetylamino galactosamine, D-Glucose aldehydic acid and L-fucose, relative molecular mass is 40,000-50,000; Another kind of is HF (Holothurian fucan) (40,41), is the straight-chain polysaccharide being made up of L-fucose, and relative molecular mass is 80,000-100,000.Though both composition glycosyls are different, have part of hydroxyl generation sulphating on sugar chain, and sulfuric ester base class polysaccharide content is all in 32% left and right, and the special construction of two kinds of sea cucumber polysaccharides is sea cucumber institute peculiar.
Sea cucumber chondroitin sulfate polysaccharide is one of two kinds of main polysaccharide of sea cucumber, its complex structure, and have difference because of the difference of sea cucumber kind and growing environment.In recent years; both at home and abroad the pharmacological action of sea cucumber chondroitin sulfate and isomer thereof is studied; prove that it has antitumor; improve immunity of organisms; antithrombotic, anticoagulation, reduces blood viscosity; neuroprotective tissue and the multiple physiologically active such as antibacterial, physiological function regulation and control to human body, maintain life optimum regime and be extremely important.
At present, the sea cucumber chondroitin sulfate of literature research report mainly contains following two kinds:
One is that Paulo A.S. etc. extracts a kind of sea cucumber polysaccharide from L.grisea sea cucumber, the structure of similar chondroitin sulfate, sugar compositional analysis shows that it contains Fuc: GalNAc: GlcA: SO4 is about 1: 1: 1: 2.7, and adopt dilute suplhuric acid hydrolysis and observe nucleus magnetic resonance in hydrolytic process in conjunction with NMR technology
1h NMR spectrogram changes and shows, Fucose side chain is mainly connected on 3 of glucuronic acid.Adopt chondroitin sulfate A (CSA) BC enzyme also product to be carried out to disaccharide composition analysis, show that main chain contains chondroitin sulfate, chondroitin sulfate 4-S and chondroitin sulfate 4,6-S.And nmr analysis shows that its side chain Fucose is with 4-O-SO
4be substituted by master, and contain 3,4 and 2,4-O-SO
4replace.
It measures its monose and sulfate composition two for the separation and purification from Apostichopus japonicus (S.japonicus) body wall such as Kariya goes out glycosaminoglycan extracted from sea cucumber after acidolysis, finds that it is chondroitin sulfate E type structure, sulfate (SO
4 2-), the mol ratio of galn (GalN), glucuronic acid (GlcUA) and Fucose (Fuc) is 3: 2: 2: 1.The Fucose side chain that further structural analysis shows this chondroitin sulfate is with 2,4-O-SO
4be main, and contain 3,4 and 4-O-SO
4replace.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of U.S.'s meat ginseng fucosylation mucopolysaccharide with antithrombotic acitivity and anticoagulant functions.
In order to solve the problems of the technologies described above, the invention provides a kind of U.S. meat ginseng fucosylation mucopolysaccharide, its structural formula is:
n＝70。
Improvement as the U.S. of the present invention meat ginseng fucosylation mucopolysaccharide: U.S.'s meat ginseng fucosylation mucopolysaccharide main chain is chondroitin sulfate E structure, the side chain simultaneously forming with sulfated fucose, this side chain is connected to by glycosidic link on β-D-Glucose aldehydic acid of main chain, and the replacement mode of sulfate is with 2,4-O-SO
4be main, for from a kind of novel structure chondroitin sulfate polysaccharide of obtaining of separating from sea cucumber.
Further improvement as the U.S. of the present invention meat ginseng fucosylation mucopolysaccharide: side chain is 2,4 Sulfated α-D-Fucoses.
Further improvement as the U.S. of the present invention meat ginseng fucosylation mucopolysaccharide: U.S.'s meat ginseng fucosylation mucopolysaccharide is the chondroitin sulfate that one contains 2,4 Sulfated α-D-Fucose side chains.
The present invention also provides the purposes of above-mentioned U.S. meat ginseng fucosylation mucopolysaccharide simultaneously: it has antithrombotic acitivity or anticoagulant functions; Therefore can be for the preparation of antithrombotic agent or anti-coagulant.
The U.S. of the present invention meat ginseng fucosylation mucopolysaccharide main chain is chondroitin sulfate E structure, the side chain simultaneously forming with sulfated fucose, this side chain is connected to by glycosidic link on β-D-Glucose aldehydic acid of main chain, and the replacement mode of sulfate is with 2,4-O-SO4 is substituted by master, for from a kind of novel structure chondroitin sulfate polysaccharide of obtaining of separating from sea cucumber.U.S.'s meat ginseng fucosylation mucopolysaccharide has the anticoagulation of comprising, antithrombotic acitivity isoreactivity.Its usage and consumption can be with reference to the usage of current existing sea cucumber chondroitin sulfate and consumptions.
In sum, the present invention finds a kind of novel fucosylation mucopolysaccharide, and the research of the anti-freezing anti-thrombus activity to it has great importance.
Brief description of the drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the infrared spectrogram of the U.S. of the present invention meat ginseng fucosylation mucopolysaccharide;
Tu2Shi U.S. meat ginseng fucosylation mucopolysaccharide
1hNMR;
The 2D spectrogram of Tu3Shi U.S. meat ginseng fucosylation mucopolysaccharide;
A; TOCSY spectrogram; B HMQC spectrogram; C NOESY spectrogram;
Tu4Shi U.S. meat ginseng fucosylation mucopolysaccharide
13cNMR;
The zymoplasm FIIa of Fig. 5 U.S. meat ginseng fucosylation mucopolysaccharide and the restraining effect of Factor Xa;
What a Antithrombin III mediated suppresses active to FIIa; B heparin factor II mediation is to suppressing active to FIIa;
What c Antithrombin III mediated suppresses active to FXa.
Embodiment
The extracting method of embodiment 1, U.S.'s meat ginseng fucosylation mucopolysaccharide, carries out following steps successively:
1), by trepang (water ratio is lower than 10%) the pulverize thing of 50g U.S. meat ginseng, add the mixing buffered soln of 1500mL, and add 5mg papoid, stirring reaction 24h under 60 DEG C of water-baths.
The preparation method of this mixing buffered soln is as follows: in every L 0.1M acetic acid-sodium acetate buffer solution (pH6.0), add the EDTA of 15mmol and the halfcystine of 5mmol.
2), by step 1) centrifugal (2000g of enzymolysis after product of gained, 15min, 20 DEG C), be 10% cetylpyridinium chloride (CPC) aqueous solution to adding 80mL mass concentration in supernatant liquor (being sea cucumber hydrolysate), under room temperature, place after 24 hours, centrifugal (2000g, 15min), abandoning supernatant.
3), by step 2) (concentration of NaCl is 3mol/L for the aqueous ethanolic solution of the NaCl that is precipitated and dissolved in 500mL of gained, ethanol water=100: 15v/v) in, add again 1000mL 95% (volumetric concentration) ethanolic soln, place 24 hours for 4 DEG C, centrifugal (2000g, 15min), precipitation uses respectively 30mL 80% and 95% (being volumetric concentration) ethanolic soln to wash 2 times, finally will be deposited in 60 DEG C and be dried 2 hours, dissolve with distilled water, be tubular fibre membrane ultrafiltration the desalination of 6000Da with the molecular weight that dams, concentrated, freeze-drying (in-62 DEG C), obtain sea cucumber Crude polysaccharides 5g, extraction yield is about 10%.
4), by sea cucumber Crude polysaccharides process DEAE-52 anionresin column separating purification.Concrete purification step is as follows:
The sea cucumber Crude polysaccharides of 3g adopts DEAE anionite-exchange resin (4.6*20cm) separation and purification, adopt the buffer salt solution (using 0.1M acetic acid-sodium acetate buffer solution (pH 6.0) as solvent) of 0-1.4mol/LNaCl to carry out linear gradient elution, overall system volume is 2000ml, flow velocity is 0.5mL/min, every 10min collects a pipe, survey light absorption value in 280nm place and detect protein content, adopt the phenolsulfuric acid method of improvement to detect the polysaccharide content of each pipe.
And by the single pipe of liquid phase TSK4000 pillar detected components, 75th～110 pipes are for meeting the pipe of above-mentioned requirements.The dialysis tubing that is 14000Da through molecular weight cut-off after collection dialysis, vacuum freezing (0.025MPa ,-62 DEG C) is dry, obtains U.S. meat ginseng fucosylation mucopolysaccharide 1.2g.
The polysaccharide of gained adopts cellulose-acetate membrane electrophoresis and high performance liquid chromatography qualification purity and molecular weight.Result shows that the molecular weight of this U.S.'s meat ginseng fucosylation mucopolysaccharide is about 109KDa.Purity is 98.2%.
(1) the U.S. about 2.0mg of meat ginseng fucosylation mucopolysaccharide that gets above-described embodiment 1 gained, in ampere bottle, adds 1mL2molL
-1tFA, inflated with nitrogen tube sealing, 110 DEG C of hydrolysis 8h.Be cooled to room temperature, 50 DEG C volatilize TFA, successively respectively with 2molL
-1, 0.3molL
-1naOH solution is adjusted to neutrality, and ultrapure water is settled to 1mL, gets the internal standard substance lactose that 400 μ L are incorporated to 50 μ L 2mM/L and carries out PMP derivatize.Chromatographic condition: chromatographic instrument: Agilent 1100 high performance liquid chromatographs, chromatographic column: ZORBAX Eclipse XDB-C18 separator column (4.6 × 150mm, 5 μ m), detector: UV-detector, 250nm, flow velocity: 1.0mLmin
-1, column temperature: 25 DEG C, moving phase: solvent orange 2 A: 15% (V/V) acetonitrile+0.05molL-1 phosphoric acid buffer (KH2PO4-NaOH, pH 6.9), solvent B:40% (V/V) acetonitrile+0.05molL-1 phosphoric acid buffer (KH2PO4-NaOH, pH 6.9), gradient mode: time gradient: 0 → 10 → 30min, concentration gradient: 0 → 8% → 20% solvent B, sampling volume: 10 μ L.Result shows: the monose of U.S.'s meat ginseng fucosylation mucopolysaccharide consists of glucuronic acid (GlcUA), galn (GalN) and Fucose (Fuc), and its ratio is about 1: 1: 1.1.
(2) U.S.'s meat of getting above-described embodiment 1 gained is joined fucosylation mucopolysaccharide (approximately 2mg), adds the dense HCl of 5mL and the dense HNO of 1.5mL
3digest approximately 2 hours to till micro-Huang, remaining a small amount of solution constant volume, to 50mL, is got to 2.5mL employing ion-chromatographic determination sulfate content.Make standard substance Criterion curve with potassium sulfate.Result shows that the sulfate of U.S.'s meat ginseng fucosylation mucopolysaccharide polysaccharide is about 34.3%.
(3) U.S.'s meat ginseng fucosylation mucopolysaccharide and the KBr that get above-described embodiment 1 gained suppress in flakes, use Vector22 type Fourier infrared spectrograph, scanning 4000～400cm
-1the spectral absorption value of wave-number range.Result as shown in Figure 1, shows that U.S.'s meat gracilis polysaccharide is at 3600-3200cm
-1all there is a wide O-H stretching vibration peak, at 2990cm
-1and 2940cm
-1for the absorption peak of Fucose methyl, at 1430cm
-1neighbouring (carboxyl C=O is flexible ,-OH bending) located absorption, illustrates in U.S.'s meat ginseng fucosylation mucopolysaccharide and contains uronic acid, and this is consistent with monose compositional analysis result.This external 1261～1220cm
-1(stretching vibration peak of S=O) and 860～820cm
-1(stretching vibration peak of C-O-S) located strong absorption, further shows that all samples is the polysaccharide that is rich in sulfate.Wherein 827cm
-1the absorption peak at place is the stretching vibration (axial coordination) of C-O-S, is the Fucose that C-4 position sulfate replaces, and illustrates that U.S.'s meat ginseng fucosylation mucopolysaccharide is with C-2, and 4 are substituted by master;
(4) 50mg respectively got in U.S.'s meat ginseng fucosylation mucopolysaccharide of above-described embodiment 1 gained, with 0.5mL D
2o (99.96%) exchanges 2 times continuously, through 0.5mL D
2after O (99.96%) dissolves, by JNM-ECP 600 nuclear magnetic resonance spectrometers mensuration
1h NMR.Condition determination: 60 DEG C, 600MHz; Interior mark: acetone.
13c NMR and two-dimentional spectrogram
1h-
1h COSY, TOCSY analyzes and obtains at 20 DEG C.For sugar compounds,
1proton (H-1) signal in H NMR spectrum on C-1, conventionally at δ 4.8～5.6ppm, is more easily resolved, and the proton signal of other C-2～C-6 all concentrates on δ 3.2～4.8ppm, and the intersection that overlaps each other is resolved difficulty.U.S.'s meat ginseng fucosylation mucopolysaccharide main chain is the structure that is similar to the chondroitin sulfate being alternately made up of β-D-GlcUA and β-D-GalNAc, and side chain is Fucose glycosyl.The signal at 5.1～5.6ppm place is attributed to the anomer hydrogen signal of Fucose.U.S. meat ginseng fucosylation mucopolysaccharide has shown the peak (Fig. 2) a little less than a main peaks and intensity are at 5.56ppm place, the data with reference to Mourao etc. in sea cucumber L.grisea
[7], they are attributed to 2,4-O-SO
4.In addition U.S.'s meat ginseng fucosylation mucopolysaccharide also has the weak peak of a signal at 5.23ppm, and data in literature before reference, is attributed to 4-O-SO
4.In the methyl signals region of Fucose (1.0～1.3ppm), U.S.'s meat ginseng fucosylation mucopolysaccharide (Fig. 2-4a) has only shown the methyl signals of 1.19ppm, therefore can be belonged to 2,4-O-SO
4methyl hydrogen signal.U.S.'s meat ginseng fucosylation mucopolysaccharide, has only shown a methyl signals peak in 1.89-1.93ppm region, show that the replacement type of GalNAc on their main chain is similar (chondroitin sulfate E below discusses).(Fig. 3 a) has shown the coherent signal (as shown in Figure 3 a) of Fucose anomer hydrogen signal H-1 and H-2 to two dimension TOCSY, if a1/a is the coherent signal (as shown in Figure 3 a) of H-1 and H-2 in 2,4 Fucoses.The chemical shift of all fucosidos is as shown in table 1.HMQC and NEOSY spectrogram have shown that its main chain is the structure (as shown in Fig. 3 b and 3c) of chondroitin sulfate E.
Table 1 U.S. meat ginseng fucosylation mucopolysaccharide fucosido
1the displacement of H nmr chemical
"-", represents that sea cucumber polysaccharide signal is not detected
5) U.S.'s meat is joined fucosylation mucopolysaccharide
13c-NMR spectrogram is as Fig. 4 institute.It show that unlike hydrogen spectrum the sulfate of sea cucumber fucosido replaces situation straightforwardly.But we can show that the carbon of U.S.'s meat ginseng fucosylation mucopolysaccharide is composed and the carbon spectrum of standard chondroitin sulfate E and stichopus japonicus S.japonicus chondroitin sulfate (its main chain is chondroitin sulfate E structure) is closely similar, and therefore first we analyze its main chain.The signal of 67.5ppm can clearly belong to the C-6 position of Sulfated GalNAc, but the signal of C-4 is owing to being stacked and being difficult to ownership with other signals.In anomeric carbon part, U.S. meat ginseng (Fig. 4) 97.1,99.8 and 104.1ppm shown three very clearly signals, belong to respectively its three monosaccharide unit Fuc, the anomeric carbon signal of GalNAc and GlcA.
6) structural formula of embodiment 1 gained U.S. meat ginseng fucosylation mucopolysaccharide is:
n～＝70。
The anti-freezing anti-thrombus activity of embodiment 3, U.S.'s meat ginseng
1), people's venous blood sampling, add in the plastics tubing containing the citric acid sodium antithrombotics of 0.109mol/L according to 9: 1 (volume ratio) ratios.Mix gently, 3000r/min, 15min is centrifugal, gets blood plasma and tests for blood coagulation activity; For prothrombin time (PT) experiment, 72 μ L blood plasma mix 8 μ L sample solutions, hatch 1min for 37 DEG C.Then give in mixed solution and add 20 μ L PT detection reagent, hatch 5min for 37 DEG C, the clotting time is noted down by automatic blood coagulation instrument simultaneously.For activated partial thromboplastin time (APTT) experiment, 72 μ L blood plasma mix 8 μ L sample solutions, hatch 1min for 37 DEG C.Then give in mixed solution and add 20 μ L APTT detection reagent, 70C is hatched 5min.Finally add pre-temperature (37 DEG C) 10 μ L, (calciumchloride solution, the clotting time is by the timing of automatic blood coagulation instrument simultaneously for 0.025mol/L calcium chloride.To thrombin time (TT) experiment, 45 μ L blood plasma mix 5 μ L sample liquid, hatch 1min for 37 DEG C, finally add 50 μ L TT detection reagent, note down the clotting time simultaneously.All blood coagulations are tested equal parallel running 6 times, get average.Anticoagulating active was represented by the clotting time.Heparin sodium is as positive drug, and the U.S. to be measured meat ginseng fucosylation mucopolysaccharide final concentration is respectively 4,16,64 μ g/mL.Blank serum compares.All samples comprises that heparin sodium is all dissolved in physiological saline.Experimental result shows, the U.S. of the present invention meat ginseng fucosylation mucopolysaccharide has and extends significantly APTT and the effect of TT time, and does not extend PT chronergy, it is active compare with standard heparin after, be respectively 183IU/mg and 157IU/mg.
2), the inhibition to zymoplasm (FIIa) and Factor Xa.This experiment is carried out at 384 hole microwell plates, reaction system final volume 40 μ L, comprising final concentration is 10nmol/L Antithrombin III (AT III) or 30nmol/L HC II, 2nmol/LFIIa or FXa, and U.S.'s meat ginseng fucosylation mucopolysaccharide and the heparin sodium sample (0.00025 μ g/mL-25 μ g/mL concentration gradient) of different concns.Each sample is all dissolved in TS/PEG buffer (0.02mol/L Tris/HCl, 0.15mol/LNaCl and 1.0mg/mL PEG 8000, pH 7.4).FIIa/FXa finally adds reaction system to start reaction, hatches 60s for 37 DEG C, then adds 25 μ L 0.4mmol/L FIIa chromophoric substrate/FXa chromophoric substrates, then microwell plate is inserted in microplate reader, records 405nm OD value 300s.OD velocity of variation and to be retained in FIIa/FXa activity in system proportional, carrys out the thrombin vigor in observe system according to the velocity of variation of OD, thereby calculates the restraining effect of the sample adding to thrombin.Blank assay comprises that FIIa/FXa mixes and hatches with AT III/HC II, but does not add each sample.In blank assay, have no the restraining effect of any thrombin.All experiment parallel runnings 3 times.
Its activity as shown in Figure 5, Fig. 5 a, along with the concentration of U.S. meat ginseng fucosylation mucopolysaccharide increases, in system, the vigor of FIIa reduces gradually, shows that its restraining effect increases gradually, but its activity is all significantly lower than the activity of heparin.Fig. 5 b is than Fig. 5 a, coagulation cofactor changes, be changed to HC II by ATIII, target protein enzyme is still F II a, and figure trend fundamental sum Fig. 5 a is consistent, along with adding the increase of sample dose, the restraining effect of sample strengthens gradually, and ATIII mediation is different, and its activity is a little more than heparin sample, in this explanation U.S. meat ginseng fucosylation mucopolysaccharide, exist HC II binding site, he is combined the activity of rear and then Trombin inhibiting with heparin factor II.Fig. 5 c has shown the effect curve of the inhibition FXa of AT III mediation, by seeing in figure that its activity is obviously weaker than standard heparin.
3), U.S.'s meat ginseng fucosylation mucopolysaccharide In Vitro Anti thrombus experiment.Healthy male SD rat, body weight 250～300g, be divided at random 5 groups by body weight, be respectively Normal group, heparin control group and low molecular weight heparin group (dosage is respectively 0.2mg/kg and 0.5mk/kg), the low and high dose group (dosage is respectively 0.3mg/kg and 0.5mg/kg) of sea cucumber meat ginseng.Each group is pressed 1mL/kg tail vein injection medicine, Normal group injecting normal saline, 1 time/d, 3d continuously.0.5h after the administration of SD rat, with 20% urethane anesthesia, abdominal aortic blood.Get blood 4mL for every, join the liquor sodii citratis 0.5mL that inserts in advance 0.38%, mix.Get 1mL anticoagulated blood and join in thrombus tube, and put in the thrombus determinator that is preheated to 37 DEG C, rotate after 10min, take off thrombus tube, thrombus wherein and blood are together poured on filter paper, by vernier caliper measurement thrombus length.Again the scraps of paper that are placed with thrombus are put in constant temperature roaster, after 60 DEG C of oven dry 40min, taken thrombus dry weight.Experimental result adopts the significant difference of Tukey ' s test evaluation result, using p < 0.05 as the standard that has significant difference.All results all represent with means ± SD.All analytic statistics inspections adopt GraphPad Instat 4.0 softwares (GraphPad Software, San Diego, CA, USA).Experiment shows, U.S.'s meat is joined fucosylation mucopolysaccharide group (high dose group (0.5/mg/ml) and low dose group (0.3/mg/ml)) compared with normal group, aobvious reduce (the P < 0.01) of its wet weight of thrombus; But its action effect is compared with low molecular weight heparin group with heparin group, the action effect of its high dose group (0.5/mg/ml) is still lower than the action effect of heparin group (0.3/mg/ml), but higher than the action effect of low molecular weight heparin group, and the action effect of low dose group (0.3/mg/ml) is suitable with low molecular weight heparin group.
The In Vitro Anti thrombus research of table 2 U.S. meat ginseng fucosylation mucopolysaccharide
| Group | Thrombus length cm | Thrombus dry weight mg |
| Normal group | 2.16±0.52 | 110.47±9.49 |
| Standard heparin control group (0.3/mg/ml) | Do not form thrombus | Do not form thrombus |
| Low molecular weight heparin group (0.5/mg/ml) | 1.83±0.74 | 35.83±4.73 * |
| MR-CHS(0.3/mg/ml) | 1.89±0.39 | 35.40±7.15 * |
| MR-CHS(0.5/mg/ml) | 1.81±0.27 | 32.73±5.35 ** |
contrast experiment 1: separation and purification goes out Apostichopus japonicus (S.japonicus) body wall sea cucumber chondroitin sulfate, study its external anticoagulant efficiency, its external prolongation APTT activity of result is 100IU/mg, TT activity is 80IU/mg, is starkly lower than U.S.'s meat ginseng fucosylation mucopolysaccharide of the present invention's report.The activation analysis of In Vitro Anti thrombus shows, when its 0.5mg/kg dosage, its external formation thrombus dry weight is 38.7 ± 6.75, and its external antithrombotic effect is weaker than polysaccharide involved in the present invention.Therefore, compared with Apostichopus japonicus chondroitin sulfate, U.S. meat ginseng fucosylation mucopolysaccharide be obviously better than Apostichopus japonicus, and in experimentation, do not find obvious hemorrhage side effect, having external antithrombotic acitivity simultaneously and be better than Apostichopus japonicus, is a kind of potential good anticoagulation and antithrombotic reagent.
Contrast experiment 2: separation and purification goes out Brazilian gulf sea cucumber (L.grisea) body wall sea cucumber chondroitin sulfate, study its external anticoagulant efficiency, its external prolongation APTT activity of result is 40IU/mg, TT activity is 32IU/mg, is starkly lower than U.S.'s meat ginseng fucosylation mucopolysaccharide of the present invention's report.The activation analysis of In Vitro Anti thrombus shows, when its 0.5mg/kg dosage, its external formation thrombus dry weight is 42.35 ± 6.75, and its external antithrombotic effect is weaker than polysaccharide involved in the present invention.Therefore, compared with the sea cucumber chondroitin sulfate of Brazilian gulf, U.S. meat ginseng fucosylation mucopolysaccharide be obviously better than Apostichopus japonicus, and in experimentation, do not find obvious hemorrhage side effect, having external antithrombotic acitivity simultaneously and be better than Apostichopus japonicus, is a kind of potential good anticoagulation and antithrombotic reagent.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (1)
1. U.S.'s meat ginseng fucosylation mucopolysaccharide, is characterized in that structural formula is:
The molecular weight of described U.S. meat ginseng fucosylation mucopolysaccharide is 109KDa, and purity is 98.2%.
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| CN103536621A (en) * | 2012-07-11 | 2014-01-29 | 上海开润生物医药有限公司 | Application of holothurian glycosaminoglycan in preparation of medicaments for treating coronary syndromes |
| CN104558223B (en) * | 2014-09-17 | 2016-10-05 | 中国海洋大学 | A kind of high-purity Apostichopus japonicus glycosaminoglycans and its preparation method and application |
| CN105651921B (en) * | 2016-01-14 | 2017-12-12 | 中国海洋大学 | A kind of method for differentiating anax on vascular endothelial using sulfated oligosaccharide group |
| CN106645483B (en) * | 2016-12-26 | 2019-05-21 | 大连工业大学 | A kind of method of quantitative detection sea cucumber polysaccharide |
| CN110437288B (en) * | 2019-09-02 | 2021-06-08 | 中国海洋大学 | Sea cucumber fucoidin and preparation method and application thereof |
| WO2022067774A1 (en) * | 2020-09-30 | 2022-04-07 | 牡丹江友搏药业有限责任公司 | Preparation method and application of sea cucumber polysaccharide |
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