CN102367285A - Pearsonothuria graeffei fucosylated chondroitin sulfate, extracting method thereof, and purpose thereof - Google Patents
Pearsonothuria graeffei fucosylated chondroitin sulfate, extracting method thereof, and purpose thereof Download PDFInfo
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- CN102367285A CN102367285A CN201110347315XA CN201110347315A CN102367285A CN 102367285 A CN102367285 A CN 102367285A CN 201110347315X A CN201110347315X A CN 201110347315XA CN 201110347315 A CN201110347315 A CN 201110347315A CN 102367285 A CN102367285 A CN 102367285A
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- stichopus japonicus
- sea cucumber
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- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 title abstract description 8
- 229920001287 Chondroitin sulfate Polymers 0.000 title abstract description 8
- 229940059329 chondroitin sulfate Drugs 0.000 title abstract description 8
- 241000960981 Pearsonothuria graeffei Species 0.000 title abstract 3
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Abstract
The invention discloses pearsonothuria graeffei fucosylated chondroitin sulfate with a structural formula represented as the following. The main chain is in a chondroitin sulfate A structure. The structure also contains a branched chain composed of fucoidan sulfate. The branched chain is connected on a beta-D-glycuronic acid of the main chain through a glucosidic bond. The substitution manners of sulfate groups are 75%3,4-O-SO4, 20%2,4-O-SO4, and 5%4-O-SO4. The invention also discloses an extraction method of pearsonothuria graeffei fucosylated chondroitin sulfate. The polysaccharide provided by the invention provides a function of anticoagulation or antithrombosis.
Description
Technical field
The present invention relates to a kind of macromolecular cpd--Philippines's stichopus japonicus CHS polysaccharide and process for extracting and purposes belong to the natural polymer field.
Background technology
Discover that the polysaccharide that is present in the sea cucumber body wall mainly divides two types: one type is the sea cucumber CHS of fucosylation (sea cucumber fucolysated chondroitin sulfate) (38; 39); By branch's mixed polysaccharide that D-N-acetylamino galactosamine, D-glucuronic acid and L-Fucose are formed, relative molecular mass is 40,000-50,000; Another kind of is sea cucumber fucoidin (Holothurian fucan) (40,41), is the straight-chain polysaccharide that is made up of the L-Fucose, and relative molecular mass is 80,000-100,000.Though both composition glycosyls are different, and part of hydroxyl generation sulphating is all arranged on the sugar chain, and sulfuric ester base class polysaccharide content is all about 32%, the special construction of two kinds of sea cucumber polysaccharides, and it is peculiar to be sea cucumber institute.
The sea cucumber CHS is one of two kinds of main polysaccharide of sea cucumber, its complex structure, and difference is arranged because of sea cucumber kind and growing environment different.In recent years, both at home and abroad the pharmacological action of sea cucumber chondroitin sulfate and isomer thereof is studied, it is antitumor to prove that it has; Enhance immunity power; Antithrombotic, anticoagulation reduces blood viscosity; Neuroprotective tissue and multiple physiologically active such as antibacterial are to the physiological function regulation and control of human body, maintain the life optimum regime and be extremely important.
At present, the sea cucumber CHS of literature research report mainly contains following two kinds:
One of which is that Paulo A.S. etc. extracts a kind of sea cucumber polysaccharide from the L.grisea sea cucumber; The structure of similar CHS; The sugar compositional analysis shows that it contains Fuc: GalNAc: GlcA: SO4 is about 1: 1: 1: 2.7, and adopt the dilute suplhuric acid hydrolysis and combine the NMR technology to observe nucleus magnetic resonance in the hydrolytic process
1H NMR spectrogram changes and to show, the Fucose side chain mainly is connected on 3 of glucuronic acid.Adopt chondroitin sulfate A (CSA) BC enzyme and product carried out the disaccharides compositional analysis, show that main chain contains CHS, CHS 4-S and CHS 4,6-S.And nmr analysis shows that its side chain Fucose is with 4-O-SO
4Be substituted by the master, and contain 3,4 and 2,4-O-SO
4Replace.
It measures its monose and sulfate composition two for separation and purification from Japanese stichopus japonicus (S.japonicus) body wall such as Kariya goes out glycosaminoglycan extracted from sea cucumber after acidolysis, find that it is a CHS E type structure, sulfate (SO
4 2-), the mol ratio of galn (GalN), glucuronic acid (GlcUA) and Fucose (Fuc) is 3: 2: 2: 1.Further structural analysis show this CHS the Fucose side chain with 2,4-O-SO
4Be main, and contain 3,4 and 4-O-SO
4Replace.
Summary of the invention
The technical problem that the present invention will solve provides a kind of Philippines stichopus japonicus CHS polysaccharide and process for extracting and purposes; This Philippines's stichopus japonicus CHS polysaccharide has anticoagulation, antithrombotic acitivity isoreactivity.
In order to solve the problems of the technologies described above, the present invention provides a kind of Philippines stichopus japonicus CHS polysaccharide, and its structural formula is following:
Improvement as Philippines of the present invention stichopus japonicus CHS polysaccharide: main chain is a CHS E structure; Have the side chain that sulfated fucose constitutes simultaneously; This side chain is connected on the β-D-glucuronic acid of main chain through glycosidic link, and the replacement mode of sulfate is 75%3,4-O-SO
4, 20%2,4-O-SO
4And 5%4-O-SO
4
Compare with the sea cucumber CHS of finding with present, Philippines of the present invention its principal character of stichopus japonicus CHS polysaccharide is that side chain contains 75%3,4-O-SO
4Replace.
The present invention provides the process for extracting of above-mentioned Philippines stichopus japonicus CHS polysaccharide simultaneously, may further comprise the steps:
1), solid sea cucumber (be water cut less than 10% Philippines stichopus japonicus, above-mentioned % is quality %) is pulverized or is cut into small pieces, adds papoid in 50～70 ℃ of enzymolysis 12～48 hours; The mass ratio of papoid and solid sea cucumber is 1: 8～12;
2), the enzymolysis after product of step 1) gained is centrifugal, get supernatant; In supernatant, add mass concentration and be 8%～12% TMBEA (CPC) solution and place after 20～28 hours, centrifugal, abandoning supernatant; Must precipitate;
The liquid material ratio of solid sea cucumber in TMBEA solution and the step 1) is: 1.4～1.8ml TMBEA solution/1g solid sea cucumber;
3), with step 2) resolution of precipitate of gained in the aqueous ethanolic solution of NaCl, add the ethanol of volumetric concentration >=95% again, placed 22～26 hours for 3～5 ℃, centrifugal, deposition is with the washing with alcohol after drying of volumetric concentration >=80%; Dry postprecipitation use dissolved in distilled water earlier, uses the molecular weight that dams to be tubular fibre membrane ultrafiltration and the desalination of 6000Da then, concentrated, freeze-drying, crude product (being the sea cucumber Crude polysaccharides) that must Philippines's stichopus japonicus CHS polysaccharide;
In the aqueous ethanolic solution of NaCl: the concentration of NaCl is 2.5～3.53mol/L, and aqueous ethanolic solution is that ethanol mixes according to 100: 10～20 volume ratio with water and gets;
The ethanol of volumetric concentration >=95% is 1～2: 1 with volume ratio in the aqueous ethanolic solution of NaCl;
The consumption of the aqueous ethanolic solution of NaCl requires can dissolving step 2) deposition of gained, be generally the aqueous ethanolic solution of the NaCl of every 1g solid sea cucumber adapted 12～17ml.
Improvement as the process for extracting of Philippines of the present invention stichopus japonicus CHS polysaccharide: with the crude product of Philippines's stichopus japonicus CHS polysaccharide through the amberlite glue purification, Philippines's stichopus japonicus CHS polysaccharide.
Further improvement as the process for extracting of Philippines of the present invention stichopus japonicus CHS polysaccharide: above-mentioned purification process is specially:
The crude product of Philippines's stichopus japonicus CHS polysaccharide is adopted DEAE anionite-exchange resin (2.6*20cm) separation and purification; Adopt 0-1.4mol/LNaCl buffer salt solution (with 0.1M acetate-sodium acetate buffer solution (pH 6.0) as solvent) to carry out gradient elution; Detect collected elutriant through liquid phase TSK4000 pillar; Is the dialysis tubing dialysis of 14000Da with the single elutriant of component through molecular weight cut-off, and vacuum lyophilization gets Philippines's stichopus japonicus CHS polysaccharide.
The present invention also provides the purposes of above-mentioned Philippines stichopus japonicus CHS polysaccharide simultaneously, it is characterized in that: be used for anticoagulation or antithrombotic.
In the step 1) of the process for extracting of Philippines of the present invention stichopus japonicus CHS polysaccharide; After the pulverizing of solid sea cucumber or being cut into small pieces; Add earlier the mixed solution formed by 0.1M acetate-sodium acetate buffer solution (pH=6.0) and EDTA and halfcystine (in this mixed solution; The concentration of EDTA is 13～17mmol/L, and the concentration of halfcystine is 4～6mmol/L), and then the adding papoid carries out enzymolysis; Above-mentioned mixed solution is 25～35ml mixed solution/1g solid sea cucumber with the liquid material ratio of solid sea cucumber.
Philippines of the present invention stichopus japonicus CHS polysaccharide has anticoagulation, and though its activity is lower than heparin, there is the little advantage of hemorrhage side effect in the antithrombotic acitivity isoreactivity.
In terms of existing technologies: the present invention mainly provide inoculation side chain fucosido replace mode and with background technology in the two kinds of polysaccharide diverse sea cucumber CHS of informing; Philippines of the present invention its side chain of stichopus japonicus CHS polysaccharide is with 3 of uniqueness, and 4-SO4 is that main replacement mode (75%) exists.Based on its structure singularity, the present invention estimates the external anti-freezing and the anti-thrombus activity of Philippines's stichopus japonicus CHS polysaccharide.Philippines of the present invention stichopus japonicus CHS polysaccharide, it has significant antithrombotic acitivity, and hemorrhage side effect is less relatively simultaneously, has the excellent development prospect.Its method of use and consumption are with existing sea cucumber CHS.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is the ir spectra of Philippines's stichopus japonicus CHS;
Fig. 2 is Philippines's stichopus japonicus CHS
1HNMR;
Fig. 3 is the COSY spectrogram of Philippines's stichopus japonicus CHS;
Fig. 4 is Philippines's stichopus japonicus CHS
13CNMR;
Fig. 5 is the zymoplasm of Philippines's stichopus japonicus CHS and the restraining effect of FXa;
The Zuo Tu representative is to the restraining effect of zymoplasm, and right figure represents the restraining effect of FXa.
Embodiment
The process for extracting of embodiment 1, Philippines's stichopus japonicus CHS, carry out following steps successively:
1), the solid, dry sea cucumber of 1g (is lower than 10% Philippines stichopus japonicus for water ratio; This % is quality %) the pulverize thing; The mixed solution of forming by 0.1M acetate-sodium acetate buffer solution (pH=6.0) and EDTA and halfcystine (in this mixed solution, the concentration of EDTA is 15mmol/L, and the concentration of halfcystine is 5mmol/L) that adds 30mL; And adding 100mg papoid, stirring reaction under 60 ℃ of water-baths (being enzymolysis) 24h.
2), with the enzymolysis after product of step 1) gained centrifugal (2000g, 15min, 20 ℃); Get supernatant, adding 1.6mL mass concentration is 10% TMBEA (CPC) aqueous solution in supernatant (being sea cucumber hydrolysate), and the room temperature held is after 24 hours; Centrifugal (2000g; 15min), abandoning supernatant must precipitate.
3), with step 2) (concentration of NaCl is 3mol/L in the aqueous ethanolic solution of the NaCl of 15mL for the resolution of precipitate of gained; Ethanol: water=100: 15v/v), add 30mL 95% (volumetric concentration) ethanolic soln again, placed 24 hours for 4 ℃; Centrifugal (2000g; 15min), deposition is respectively cleaned 1 time with 80% ethanolic soln of 30mL and 95% (being volumetric concentration) ethanolic soln of 30mL, will be deposited in 60 ℃ of dryings at last 2 hours; With above-mentioned dry postprecipitation with dissolved in distilled water (consumption of zero(ppm) water only need can dissolve above-mentioned dry postprecipitation get final product); Use tubular fibre membrane ultrafiltration and the desalination of molecular weight of damming then as 6000Da; Concentrate; Freeze-drying (in-62 ℃), crude product (the being the sea cucumber Crude polysaccharides) 100mg of Philippines's stichopus japonicus CHS polysaccharide, extraction yield is 10%.
4), with sea cucumber Crude polysaccharides process DEAE-52 anionresin column separating purification.Concrete purification step is following:
The sea cucumber Crude polysaccharides of 1g adopts DEAE anionite-exchange resin (2.6*20cm) separation and purification; Adopt the buffer salt solution (with 0.1M acetate-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, and flow velocity is 0.5mL/min, and every 10min collects a pipe; Survey light absorption value in the 280nm place and detect protein content, adopt the polysaccharide content of each pipe of phenolsulfuric acid method detection of improvement.
And through the single pipe of liquid phase TSK4000 pillar detected components, the 60th～100 pipe is for meeting the pipe of above-mentioned requirements.Collect after molecular weight cut-off is the dialysis tubing dialysis of 14000Da, vacuum freezing (0.025MPa ,-62 ℃) drying gets Philippines's stichopus japonicus CHS polysaccharide.
Gained Philippines stichopus japonicus CHS polysaccharide adopts cellulose-acetate membrane electrophoresis and performance liquid chromatography to identify purity and molecular weight.The result shows that the molecular weight of Philippines's stichopus japonicus CHS is about 73KDa.Purity is 95.2%.
The structural formula of this Philippines's stichopus japonicus CHS is:
1), gets the about 2.0mg of polysaccharide sample in the ampere bottle, adding 1mL 2molL
-1TFA, inflated with nitrogen tube sealing, 110 ℃ of difference hydrolysis 8h.Be cooled to room temperature, 50 ℃ volatilize TFA, one by one respectively with 2molL
-1, 0.3molL
-1NaOH solution transfers to neutrality, and ultrapure water is settled to 1mL, gets 400 μ L and 50 μ L internal standard substance lactose and carries out the 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 ℃, moving phase: solvent orange 2 A: 15% (V/V) acetonitrile+0.05molL-1 phosphoric acid buffer (KH
2PO
4-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.
The result shows that its monose consists of glucuronic acid (GlcUA), galn (GalN) and Fucose (Fuc), and its mol ratio is about 1: 1: 1.1.
2), get an amount of sample of sea cucumber polysaccharide (approximately 2mg), add dense HCl of 5mL and the dense HNO of 1.5mL
3Digest about 2 hours till little Huang, remaining small volume of solution constant volume to 50mL, is got 2.5mL employing ion-chromatographic determination sulfate content.Set up typical curve with vitriolate of tartar as standard substance.The result shows that the sulfate of Philippines's stichopus japonicus CHS polysaccharide is about 27.12%.
3), exsiccant Philippines stichopus japonicus CHS polysaccharide sample and KBr are suppressed in flakes use Vector 22 type Fourier infrared spectrographs, scanning 4000～400cm
-1The spectral absorption value of wave-number range.The result shows that Philippines's stichopus japonicus sea cucumber CHS polysaccharide (Fig. 1) is at 3600-3200cm
-1A wide O-H stretching vibration peak has all appearred, at 2990cm
-1And 2940cm
-1For the absorption peak of Fucose methyl, at 1430cm
-1Near (carboxyl C=O flexible ,-OH crooked) located absorption, explain in Philippines's stichopus japonicus CHS and contain uronic acid that this and monose compositional analysis result are consistent.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, shows that further all samples is the polysaccharide that is rich in sulfate.848cm wherein
-1The absorption peak at place is the stretching vibration (axial coordination) of C-O-S, is the substituted Fucose of C-4 position sulfate, explains that Philippines's stichopus japonicus CHS is substituted by the master with C-4.
4), Philippines's stichopus japonicus CHS polysaccharide sample respectively gets 50mg, with 0.5mL D
2O (99.96%) exchanges 2 times continuously, through 0.5mL D
2After O (99.96%) dissolving, measure with JNM-ECP 600 nuclear magnetic resonance spectrometers
1H NMR.Condition determination: 60 ℃, 600MHz; Interior mark: acetone.
13C NMR and two-dimentional spectrogram
1H-
1H COSY, TOCSY analyze down at 20 ℃ and obtain.For sugar compounds,
1Proton (H-1) signal in the HNMR spectrum on the C-1 is prone to resolve usually at δ 4.8～5.6ppm, 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.Philippines's stichopus japonicus CHS main chain is the structure that is similar to the CHS that alternately is made up of β-D-GlcUA and β-D-GalNAc, and side chain is the Fucose glycosyl.5.1 the signal at～5.6ppm place is belonged to the anomer hydrogen signal for Fucose.Philippines's stichopus japonicus CHS has shown a main peaks and the more weak peak (Fig. 2) of intensity at 5.17ppm and 5.23ppm place, with reference to data in sea cucumber L.grisea such as Mourao
[7], they are 3,4 and 4-O-SO by ownership
4Philippines stichopus japonicus CHS also has a peak that signal is more weak at 5.56ppm in addition, data in literature before the reference, and being belonged to is 2,4-O-SO
4Methyl signals zone (1.0～1.3ppm) at Fucose; Philippines stichopus japonicus CHS (Fig. 2) has shown 1.23ppm and two methyl signals of 1.19ppm; The ratio of ratio and two anomer hydrogen signal 5.17ppm and 5.56ppm is approximate, therefore can be belonged to 4-O-and 2,4-O-SO
4The methyl hydrogen signal.Philippines's stichopus japonicus CHS has only shown in the 1.89-1.93ppm zone to show that the replacement type of GalNAc on their main chain is similar in a methyl signals peak.Two dimension COSY has shown the coherent signal (as shown in Figure 3) of Fucose anomer hydrogen signal H-1 and H-2, and like a1/a2, b1/b2, c1/c2 are respectively 2,4,4 and 3,4-O-SO
4The coherent signal of H-1 and H-2 (as shown in Figure 3) in the Fucose.TOCSY has further provided the residual signal that COSY can not belong to, like H-3, and H-4 and H-6 (shown in the table 1).The chemical shift of all fucosidos is as shown in table 1.
5), Philippines stichopus japonicus CHS polysaccharide
13C-NMR spectrogram such as Fig. 4 institute.It replaces situation unlike the sulfate that the hydrogen spectrum draws the sea cucumber fucosido equally straightforwardly.But we can find out that the carbon of Philippines's stichopus japonicus CHS is composed and the carbon spectrum of standard CHS E and stichopus japonicus S.japonicus CHS (its main chain is a CHS E structure) is closely similar, so we at first analyze its main chain.67.5ppm signal can clearly belong to the C-6 position of Sulfated GalNAc, but the signal of C-4 is difficult to ownership owing to be stacked with other signals.。In the anomeric carbon part, Philippines stichopus japonicus (Fig. 2) 99.1,99.8 and 104.1ppm shown three very clearly signals, belong to its three monosaccharide unit Fuc respectively, the anomeric carbon signal of GalNAc and GlcA.
Table 1, different sea cucumber CHS fucosidos
1The displacement of H nmr chemical
| fucose?standard a | 4- |
2，4-di-O-S | 3，4-di-O-S | |
| H-1 | 5.20 | 5.23 | 5.56 | 5.17 |
| H-2 | 3.76 | 3.71 | 4.36 | 3.94 |
| H-3 | 3.86 | 3.90 | 4.05 | 4.34 |
| H-4 | 3.81 | 4.56 | 4.68 | 4.89 |
| H-5 | 4.20 | -b | 4.38 | -b |
| H-6 | 1.21 | 1.23 | 1.19 | 1.19 |
The anti-freezing anti-thrombus activity of experiment 2, Philippines's stichopus japonicus CHS polysaccharide
1), people's vein gets blood, add in the plastics tubing of the structure rafter acid sodium antithrombotics that contains 0.109mol/L according to 9: 1 ratios.Mix gently, 3000r/min, 15min is centrifugal, gets serum and is used for the blood coagulation activity experiment for the PT experiment, and 72 μ L blood plasma mix 8 μ L sample solutions, hatch 1min for 37 ℃.Give then to add 20 μ L APTT detection reagent in the mixed solution, hatch 5min for 37 ℃, the clotting time is noted down by the automatic blood coagulation appearance simultaneously.For the APTT experiment, 72 μ L blood plasma mix 8 μ L sample solutions, hatch 1min for 37 ℃.Give then to add 20 μ L APTT detection reagent in the mixed solution, 70C is hatched 5min.Add temperature (37 ℃) 10 μ L in advance at last, (calciumchloride solution, the clotting time is by the timing of automatic blood coagulation appearance simultaneously for 0.025mol/L calcium chloride.To the TT experiment, 45 μ L blood plasma mix 5 μ L sample liquid, hatch 1min for 37 ℃, add 50 μ LTT detection reagent at last, 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 Philippines to be measured stichopus japonicus CHS polysaccharide final concentration is respectively 4,16,64 μ g/mL.Blank serum compares.All samples comprises that heparin sodium all is dissolved in the saline water.Experimental result shows that Philippines's stichopus japonicus chondroitin sulfate have and prolongs APTT and the effect of TT time significantly, and does not prolong the PT chronergy, and its activity is respectively 35IU/mg and 78IU/mg after comparing with standard heparin.
2), to the inhibition of zymoplasm and thrombin FXa.This experiment is carried out at 384 hole microwell plates, reaction system final volume 40 μ L, and comprising final concentration is 10nmol/L AT III or 30nmol/L HC II, 2nmol/L FIIa or FXa, and two all polysaccharide fCS-Pg and heparin sodium samples of different concns.Each sample all is dissolved among the TS/PEG buffer (pH 7.4 for 0.02mol/L Tris/HCl, 0.15mol/L NaCl and 1.0mg/mL PEG 8000).FIIa/FXa adds reaction system at last and starts reaction, hatches 60s for 37 ℃, adds 25 μ L 0.4mmol/L FIIa chromophoric substrate/FXa chromophoric substrates then, then microwell plate is inserted in the ELIASA record 405nm OD value 300s.OD velocity of variation and the FIIa/FXa activity that is retained in the system are proportional, come the thrombin vigor in the observation system according to the velocity of variation of OD, thereby calculate the sample that the adds restraining effect to thrombin.Blank assay comprises that FIIa/FXa mixes with AT III/HC II hatches, but does not add each sample.Do not see the restraining effect of any thrombin in the blank assay.All experiment parallel runnings 3 times.Its activity is as shown in Figure 5, Fig. 5 A, and along with the concentration increase of Philippines's stichopus japonicus CHS, the vigor of FIIa reduces gradually in the system, show that its restraining effect increases gradually, but its activity all significantly is lower than the activity of heparin.Fig. 5 B is than Fig. 5 A, and coagulation cofactor changes, and is changed to HC II by ATIII; The target protein enzyme is still F II a, and figure trend fundamental sum Fig. 5 A is consistent, along with the increase that adds sample dose; The restraining effect of sample strengthens gradually, is that its activity is a little more than the heparin sample with the different of ATIII mediation; Exist HC II binding site in this explanation sea cucumber polysaccharide, he combines the activity of back and then Trombin inhibiting with heparin factor II.
3), the external antithrombotic experiment of Philippines stichopus japonicus CHS polysaccharide.Healthy male SD rat; Body weight 250～300g; Be divided into 7 groups at random by body weight, be respectively normal control group, heparin control group and low molecular weight heparin group (dosage is respectively 0.2mg/kg and 0.5mk/kg), Philippines's stichopus japonicus CHS is low and high dose group (dosage is respectively 0.3mg/kg and 0.5mg/kg).Each group is pressed 1mL/kg tail vein injection medicine, normal control group injecting normal saline, 1 time/d, continuous 3d.0.5h after the administration of SD rat, the urethane anesthesia with 20%, abdominal aortic blood.Get blood 4mL for every, join and insert 0.38% liquor sodii citratis 0.5mL in advance, mixing.Get the 1mL anticoagulated blood and join in the thrombus tube, and put in the thrombus determinator that is preheated to 37 ℃, rotate 10min after, take off thrombus tube, wherein thrombus and blood together is poured on the filter paper, with vernier caliper measurement thrombus length.The scraps of paper that will be placed with thrombus are again put in the constant temperature roaster, behind 60 ℃ of oven dry 40min, take by weighing the thrombus dry weight.Experimental result adopts the significant difference of Tukey ' s test evaluation result, with p＜0.05 as the standard that significant difference is arranged.All results all represent with means ± SD.All analytic statistics check employing GraphPad Instat 4.0 softwares (GraphPad Software, San Diego, CA, USA).Specifically as shown in table 2.Experiment shows, normal group is compared with Philippines stichopus japonicus CHS group, and the wet weight of thrombus of sea cucumber polysaccharide group shows and reduces (P＜0.01); Experiment shows that normal group is compared with sea cucumber CHS group, apparent reduce (P＜0.01) of the wet weight of thrombus of sea cucumber polysaccharide group; But its action effect is compared with heparin, and the action effect of the former high dose group still is lower than the action effect of heparin group, and still different with heparin group is that Philippines's stichopus japonicus CHS group does not have tangible hemorrhage side effect.
The external antithrombotic research of table 2 Philippines stichopus japonicus CHS
| Group | Thrombus length cm | Thrombus dry weight mg |
| The normal control group | 2.16±0.52 | 110.47±9.49 |
| The standard heparin control group | Do not form thrombus | Do not form thrombus |
| The low molecular weight heparin group | 1.83±0.74 | 35.83±4.73 * |
| FLB-CHS(0.3/mg/ml) | 1.89±0.39 | 39.70±3.53 * |
| FLB-CHS(0.5/mg/ml) | 1.81±0.27 | 36.73±6.71 * |
The contrast experiment 1: separation of pure dissolves Japanese stichopus japonicus (S.japonicus) body wall sea cucumber CHS, studies its external anticoagulant efficiency, and its external prolongation APTT activity is 100IU/mg as a result, and the TT activity is 80IU/mg, apparently higher than sea cucumber polysaccharide provided by the invention.But external antithrombotic acitivity analysis revealed, during its 0.5mg/kg dosage, its external formation thrombus dry weight is 42.7 ± 6.75, its external antithrombotic effect is weaker than polysaccharide involved in the present invention.Therefore; Compare with Japanese stichopus japonicus CHS; Philippines of the present invention stichopus japonicus CHS polysaccharide hemorrhage side effect is less, and external antithrombotic acitivity is superior to Japanese stichopus japonicus, shows that Philippines of the present invention stichopus japonicus CHS polysaccharide is the good antithrombotic reagent of a kind of potential.
The contrast experiment 2: separation of pure dissolves Brazilian gulf sea cucumber (L.grisea) body wall sea cucumber CHS, studies its external anticoagulant efficiency, and its external prolongation APTT activity is 40IU/mg as a result, and the TT activity is 32IU/mg.But external antithrombotic acitivity analysis revealed, during its 0.5mg/kg dosage, its external formation thrombus dry weight is 45.35 ± 6.75, its external antithrombotic effect is weaker than polysaccharide involved in the present invention.Therefore; Compare with Brazilian gulf sea cucumber CHS; The TT activity of Philippines of the present invention stichopus japonicus CHS polysaccharide is superior to western gulf sea cucumber CHS; And external antithrombotic acitivity is superior to western gulf sea cucumber CHS, shows that Philippines of the present invention stichopus japonicus CHS polysaccharide is the good antithrombotic reagent of a kind of potential.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (6)
1. Philippines stichopus japonicus CHS polysaccharide is characterized in that its structural formula is following:
2. Philippines according to claim 1 stichopus japonicus CHS polysaccharide; It is characterized in that: main chain is a CHS E structure; Have the side chain that sulfated fucose constitutes simultaneously; This side chain is connected on the β-D-glucuronic acid of main chain through glycosidic link, and the replacement mode of sulfate is 75%3,4-O-SO
4, 20%2,4-O-SO
4And 5%4-O-SO
4
3. according to claim 1 or claim 2 the process for extracting of Philippines's stichopus japonicus CHS polysaccharide is characterized in that may further comprise the steps:
1), the solid sea cucumber is pulverized or is cut into small pieces, adds papoid in 50～70 ℃ of enzymolysis 12～48 hours; The mass ratio of said papoid and solid sea cucumber is 1: 8～12;
2), the enzymolysis after product of step 1) gained is centrifugal, get supernatant; In supernatant, add mass concentration and be 8%～12% TMBEA solution and place after 20～28 hours, centrifugal, abandoning supernatant; Must precipitate;
The liquid material ratio of solid sea cucumber in said TMBEA solution and the step 1) is: 1.4～1.8ml TMBEA solution/1g solid sea cucumber;
3), with step 2) resolution of precipitate of gained in the aqueous ethanolic solution of NaCl, add the ethanol of volumetric concentration >=95% again, placed 22～26 hours for 3～5 ℃, centrifugal, deposition is with the washing with alcohol after drying of volumetric concentration >=80%; Dry postprecipitation use dissolved in distilled water earlier, uses the molecular weight that dams to be tubular fibre membrane ultrafiltration and the desalination of 6000Da then, concentrated, freeze-drying, crude product that must Philippines's stichopus japonicus CHS polysaccharide;
In the aqueous ethanolic solution of said NaCl: the concentration of NaCl is 2.5～3.53mol/L, and said aqueous ethanolic solution is that ethanol mixes according to 100: 10～20 volume ratio with water and gets;
The ethanol of said volumetric concentration >=95% is 1～2: 1 with volume ratio in the aqueous ethanolic solution of NaCl.
4. the process for extracting of Philippines according to claim 3 stichopus japonicus CHS polysaccharide is characterized in that:
With the crude product of Philippines's stichopus japonicus CHS polysaccharide through the amberlite glue purification, Philippines's stichopus japonicus CHS polysaccharide.
5. the process for extracting of Philippines according to claim 4 stichopus japonicus CHS polysaccharide, it is characterized in that: said purification process is:
The crude product of Philippines's stichopus japonicus CHS polysaccharide is adopted DEAE anionite-exchange resin (2.6*20cm) separation and purification; Adopt the 0-1.4mol/LNaCl buffer salt solution to carry out gradient elution; Detect collected elutriant through liquid phase TSK4000 pillar; Is the dialysis tubing dialysis of 14000Da with the single elutriant of component through molecular weight cut-off, and vacuum lyophilization gets Philippines's stichopus japonicus CHS polysaccharide.
6. according to claim 1 or claim 2 the purposes of Philippines's stichopus japonicus CHS polysaccharide is characterized in that: be used for anticoagulation or antithrombotic.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104413329A (en) * | 2013-08-22 | 2015-03-18 | 青岛蓝农谷农产品研究开发有限公司 | Chondroitin sulfate health product |
| CN104558223A (en) * | 2014-09-17 | 2015-04-29 | 中国海洋大学 | High-purity apostichopus japonicas glycosaminoglycan as well as preparation method and application thereof |
| CN105651921A (en) * | 2016-01-14 | 2016-06-08 | 中国海洋大学 | Method for identifying thelenata anax by means of sulfated oligosaccharide combination |
| CN106666247A (en) * | 2017-03-23 | 2017-05-17 | 威海金牌生物科技股份有限公司 | Foraging feed of special compound feed for breeding stichopus japonicus and preparation method of foraging feed |
| CN109251255A (en) * | 2017-07-12 | 2019-01-22 | 中国海洋大学 | A kind of novel fucosylated chondroitin sulfate FCShmAnd its preparation method and application |
| CN111187369A (en) * | 2020-03-03 | 2020-05-22 | 大连海洋大学 | Method for separating and purifying sea cucumber chondroitin sulfate by simulated moving bed chromatography |
| 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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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104413329A (en) * | 2013-08-22 | 2015-03-18 | 青岛蓝农谷农产品研究开发有限公司 | Chondroitin sulfate health product |
| CN104558223A (en) * | 2014-09-17 | 2015-04-29 | 中国海洋大学 | High-purity apostichopus japonicas glycosaminoglycan as well as preparation method and application thereof |
| 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 |
| CN105651921A (en) * | 2016-01-14 | 2016-06-08 | 中国海洋大学 | Method for identifying thelenata anax by means of sulfated oligosaccharide combination |
| CN106666247A (en) * | 2017-03-23 | 2017-05-17 | 威海金牌生物科技股份有限公司 | Foraging feed of special compound feed for breeding stichopus japonicus and preparation method of foraging feed |
| CN109251255A (en) * | 2017-07-12 | 2019-01-22 | 中国海洋大学 | A kind of novel fucosylated chondroitin sulfate FCShmAnd its preparation method and application |
| CN109251255B (en) * | 2017-07-12 | 2020-01-07 | 中国海洋大学 | Fucosylated chondroitin sulfate FCShmAnd preparation method and application thereof |
| CN111187369A (en) * | 2020-03-03 | 2020-05-22 | 大连海洋大学 | Method for separating and purifying sea cucumber chondroitin sulfate by simulated moving bed chromatography |
| 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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