Fuc3S4S substituted oligoglycosaminoglycan and preparation method thereof

Abstract

Disclosed are a Fuc3S4S substituted oligoglycosaminoglycan with a weight-average molecular weight (Mw) of about 4.5-9 kD, a pharmaceutical composition containing the Fuc3S4S substituted oligoglycosaminoglycan, a preparation method thereof and a use thereof in preparing medicines for preventing and/or treating thrombotic diseases.

Claims

1. A Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof, the Fuc3S4S is 3,4-disulfated-L-fucose-1-yl, the Fuc3S4S substituted oligoglycosaminoglycan mixture is a mixture of oligomeric homologous glycosaminoglycan compounds having a structure represented by Formula (I), ##STR00007## in Formula (I): Ring A is -D-glucuronic acid group or -D-glucosyl group, wherein, R1 is COO.sup. or COR.sub.10, R.sub.10 is independently substituted or unsubstituted linear or branched C1-C6 alkyl, C7-C12 aralkyl; Ring B is substituted -D-2-amino-2-deoxy-galactosyl, wherein, R.sub.2 is COCH.sub.3 or H; R.sub.3 and R.sub.4 are independently H or SO.sub.3.sup.; Ring C is -L-fucosyl, wherein, R.sub.5, R.sub.6 and R.sub.7 are independently H or SO.sub.3.sup., and based on molar ratio, the -L-fucosyl wherein R.sub.5 is H, R.sub.6 and R.sub.7 are SO.sub.3.sup., i.e. 3,4-disulfated-L-fucose-1-yl, accounts for not less than 75% of the total -L-fucosyl; R8 is the structure represented by Formula (III): ##STR00008## in Formula (III): Ring A is 4-deoxy-4-threo-hex-4-enopyranosyluronic acid group, R.sub.1 in the formula is defined as above; Ring C is -L-fucosyl, wherein, R.sub.5, R.sub.6 and R.sub.7 are defined as above; R9 is a structure represented by Formula (IV) or Formula (V): ##STR00009## in Formula (IV) and (V), Ring B is substituted a or -D-2-amino-2-deoxy-galactosyl, B is substituted 2-amino-2-deoxy-galactitol, glycosamine or N-substituted glucosamine, wherein, R.sub.3 and R.sub.4 are defined as above; R.sub.11 is hydroxy, amino, C1-C6 alkylamino, C7-C12 arylamino; n is an integer of 2-20; and based on molar ratio, the compound that n is 4-9 accounts for not less than 75% of the total compounds; the oligomeric homologues glycosaminoglycan mixture has a weight average molecular weight (Mw) of 4.5-9 kD, and a polydispersity index (PDI) of less than or equal to 1.6, wherein the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof is produced by deacylated deaminated depolymerization or -eliminative depolymerization.

2. The Fuc3S4S substituted oligoglycosaminoglycan mixture or pharmaceutically acceptable salt thereof of claim 1, wherein the mixture is a mixture of oligomeric homologues glycosaminoglycan compounds having a structure represented by Formula (VII), ##STR00010## in Formula (VII): chemical structure fragments A, A, B, B, C are defined as in claim 1; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.11 are defined as in claim 1; n is an integer of 3-15, based on molar ratio, the compounds which n is 4-9 account for not less than 75% of the total compounds; based on molar ratio, in the compounds of Formula (VII), L-fucose-3,4-disulfated, i.e. -L-Fuc3S4S group, accounts for not less than 75% of the total -L-fucosyl; and the oligomeric homologues glycosaminoglycan mixture has a weight-average molecular weight (Mw) of 4.5-9 kD.

3. The Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof according to claim 1, wherein the oligoglycosaminoglycan mixture is prepared by chemical depolymerization of Fuc3S4S substituted glycosaminoglycan from sea cucumber, wherein the Fuc3S4S substituted glycosaminoglycan has the following features: monosaccharide composition comprises D-glucuronic acid, D-acetylgalactosamine and L-fucose in a molar ratio of 1:(10.3):(10.3); based on molar ratio, in the contained -L-fucosyl, the proportion of 3,4-disulfated--L-fucosyl is no less than 75%.

4. The Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof of claim 3, wherein the Fuc3S4S substituted glycosaminoglycan from sea cucumber is obtained by extracting and purifying fresh or dried body wall and/or viscus of Holothuria scabra or Pearsonothuria graeffei.

5. The Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof of claim 1, wherein the pharmaceutically acceptable salt is sodium, potassium or calcium salt of the Fuc3S4S substituted oligoglycosaminoglycan mixture.

6. The Fuc3S4S substituted oligoglycosaminoglycan mixture or pharmaceutically acceptable salt thereof of claim 1, wherein the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof is produced without a step of carboxyl reduction with a carbodiimide compound.

7. The Fuc3S4S substituted oligoglycosaminoglycan mixture or pharmaceutically acceptable salt thereof of claim 1, wherein the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof is produced without a step of carboxyl reduction with a carbodiimide compound and a reducing agent.

8. The Fuc3S4S substituted oligoglycosaminoglycan mixture or pharmaceutically acceptable salt thereof of claim 1, wherein the at least one Ring A is a -D-glucuronic acid group, wherein, R.sub.1 is COO.

9. The Fuc3S4S substituted oligoglycosaminoglycan mixture or pharmaceutically acceptable salt thereof of claim 1, wherein Ring A is devoid of a D-glucosyl.

10. A pharmaceutical composition comprising the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof of claim 1, wherein the pharmaceutical composition comprises an anti-clotting effective amount of the oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient and/or a pharmaceutical adjuvant.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is in a dosage form of an aqueous solution for injection or freeze-dried powder for injection.

12. The pharmaceutical composition of claim 11, wherein the adjuvant of the pharmaceutical composition is a pharmaceutically acceptable sodium chloride, phosphate buffer.

13. The pharmaceutical composition of claim 10, wherein the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof is produced without a step of carboxyl reduction with a carbodiimide compound.

14. The pharmaceutical composition of claim 10, wherein the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof is produced without a step of carboxyl reduction with a carbodiimide compound and a reducing agent.

15. The pharmaceutical composition of claim 10, wherein the at least one Ring A is a -D-glucuronic acid group, wherein, R.sub.1 is COO.

16. The pharmaceutical composition of claim 10, wherein Ring A is devoid of a D-glucosyl.

17. A method of preparing the Fuc3S4S substituted oligoglycosaminoglycan mixture or pharmaceutically acceptable salt thereof of claim 1, the method comprising: obtaining a polysaccharide composition containing a Fuc3S4S substituted glycosaminoglycan; performing quaternary ammonium transalification; performing carboxyl esterification; performing -eliminative depolymerization; and performing purification to obtain the Fuc3S4S substituted oligoglycosaminoglycan mixture or a pharmaceutically acceptable salt thereof, wherein the Fuc3S4S is 3,4-disulfated-L-fucose-1-yl, wherein the Fuc3S4S substituted oligoglycosaminoglycan mixture is a mixture of the oligomeric homologous glycosaminoglycan compounds, the method being performed without a step of carboxyl reduction with a carbodiimide compound.

18. A method for treating thrombotic diseases, comprising administering to a patient in need of a therapeutically or prophylactically effective amount of the pharmaceutical composition of claim 10 to provide the anti-clotting effect.

Description

DESCRIPTION OF THE INVENTION

(1) FIG. 1. HPGPC spectrum of polysaccharide composition containing Fuc3S4S substituted glycosaminoglycan from Holothuria scabra

(2) FIG. 2. .sup.1H/.sup.13C NMR spectra of Fuc3S4S substituted oligoglycosaminoglycan mixture dHSG-1

(3) FIG. 3. (partial) superposition of .sup.1H-.sup.1H COSY, ROESY and TOCSY spectra of dHSG-1

(4) FIG. 4. .sup.1H/.sup.13C NMR spectra of Fuc3S4S substituted oligoglycosaminoglycan mixture dHSG-3

(5) FIG. 5. (partial) superposition of .sup.1H-.sup.1H COSY, ROESY and TOCSY spectra of dHSG-3

(6) FIG. 6. .sup.1H-NMR spectra of dFGAGs from different sea cucumbers

(7) FIG. 7. Effect of FGAG and dFGAG from different species on f.XII activity

(8) FIG. 8. Effect of HSG and dHSG with series of molecular weights on f.Xase activity

DETAILED DESCRIPTION OF THE INVENTION

(9) The present invention is described in detail below with reference to certain specific examples, but these specific examples do not limit the scope claimed in the present invention in any way.

Example 1

Extraction and Purification of Polysaccharide Composition Containing Fuc3S4S Substituted Glycosaminoglycans from Holothuria scabra (HSG-1, HSG-1, HSG-3)

(10) 1.1 Materials

(11) Raw Material:

(12) dried Holothuria scabra, commercially available.

(13) Reagents:

(14) papain, 810.sup.5 U/g, Nanning Pangbo Biological Engineering Co., LTD (Guangxi). NaOH, KCOCH.sub.3, H.sub.2O.sub.2 and ethanol and others were commercially available analytical reagents.

(15) 1.2 Extraction and Purification

(16) Extraction:

(17) 1000 g of dried Holothuria scabra was mechanically sliced, grounded and placed in a round-bottomed flask. 10 L of water was added to soak it overnight. The water bath was heated to 50 C., and 5 g of papain was added and fully mixed, and stirred at 50 C. for 6 hr for digestion. Subsequently, solid sodium hydroxide was added to a concentration of about 0.5 M, and alkaline hydrolysis was performed at 60 C. for 2 hr. Then, 6 N hydrochloric acid was used to adjust pH to 6-7, the water bath was cooled to room temperature, centrifuged at 4000 rpm15 min. The supernatant was added with ethanol to a final concentration of 70% (v/v), placed at 4 C. overnight, centrifuged to obtain the precipitate (crude polysaccharide from Holothuria scabra).

(18) Purification:

(19) The obtained crude polysaccharide from Holothuria scabra was dissolved in 2 L pure water, centrifuged at 4000 rpm15 min and the insoluble substances were removed. The supernatant was adjusted with 2N NaOH to pH about 9.5, and H.sub.2O.sub.2 was added to a final concentration of about 3% (v/v), reacted at 50 C. for 2 hr under stirring and subjected to decolorization. Potassium acetate was added to the decolored reaction solution to a concentration of about 0.5 M, and ethanol was added to a concentration of about 40% (v/v), placed for 4 hr at room temperature, centrifuged at 4000 rpm15 min. The precipitate was dissolved in 1 L pure water, potassium acetate was added to the obtained solution to a concentration of about 0.5 M, and ethanol was added to a concentration of about 40% (v/v), placed for 4 hr at room temperature, centrifuged at 4000 rpm15 min. The precipitate was washed with 200 mL of 80% ethanol (v/v) twice, dried under reduced pressure, and about 13.26 g of polysaccharide composition 1 containing Fuc3S4S substituted glycosaminoglycan from Holothuria scabra (HSG-1) was got.

(20) About 6.0 g of polysaccharide composition from Holothuria scabra (HSG-1) was dissolved in 500 mL of pure water, ultrafiltrated with a 100 kD ultrafiltration package using a Minipole ultrafiltration device. The resulting filtrate was ultrafiltrated with a 30 kD ultrafiltration package with the same device, and the retentate was freeze-dried to obtain about 5.33 g of polysaccharide composition 2 containing Fuc3S4S substituted glycosaminoglycan from Holothuria scabra (HSG-2).

(21) Another about 4.0 g of polysaccharide composition 1 (HSG-1) from Holothuria scabra was dissolved in 50 mL of pure water, loaded onto a DEAE-cellulose column (diameter 5 cm, bed volume 400 mL), washed with 400 mL of water (flow rate 4 ml/min), eluted with NaCl gradient from 0.5 M to 2 M. The eluted fractions were collected using an automatical collecting instrument at a rate of 10 ml/fraction. Azure solution metachromatic detection method was used to monitor the fractions containing acidic mucopolysaccharides. The fractions containing acidic mucopolysaccharides were combined and dialyzed with a 30 kD dialysis membrane, and freeze-dried to obtain about 3.25 g of polysaccharide composition 3 containing Fuc3S4S substituted glycosaminoglycan from Holothuria scabra (HSG-3).

(22) 1.3 Detection and Analysis

(23) (1) High Performance Gel Chromatography (HPGPC) Analysis:

(24) Each of the Fuc3S4S substituted glycosaminoglycan-containing polysaccharide compositions from Holothuria scabra, i.e., HSG-1, HSG-2 and HSG-3 was prepared into 5 mg/ml sample solution with pure water. The HPGPC analysis conditions were: Agilent 1200 high performance liquid chromatography meter, Shodex SB-804 HQ (8.0 mm ID300 mm) chromatography column, column temperature 35 C., injection volume 20 l, mobile phase 0.1 M NaCl solution, flow rate 0.5 ml/min, and measurement by RID detector and DAD detector.

(25) HPGPC analysis spectra of polysaccharide compositions from Holothuria scabra, i.e., HSG-1, HSG-2 and HSG-3, are shown in FIG. 1. It can be seen from FIG. 1 that HSG-2 has polysaccharide composition only at the retention time (RT) of about 19 min (mainly FGAG); besides the main peak, HSG-1 also had peaks at the retention time (RT) of about 14 min (mainly FS) and RT of about 22 min (mainly peptide ingredients); except the main peak, the peaks of HSG-3 at RT of about 14 min and RT of about 22 min were lower than that of HSG-1.

(26) (2) Molecular Weight Detection:

(27) Each of HSG-1, HSG-2 and HSG-3 and FGAG reference with series of molecular weights (prepared by the Kunming Institute of Botany, calibrated by HPLC-LALLS) was prepared into sample solution with a concentration of 10 mg/ml using pure water, and HPGPC spectra were measured.

(28) The HPGPC detection conditions were: Agilent 1200 high performance liquid chromatography meter, Shodex SB-804 HQ (8.0 mm ID300 mm) chromatography column, column temperature 35 C., injection volume 20 l, mobile phase 0.1M NaCl solution, flow rate 0.5 ml/min, and measurement by RID detector and DAD detector.

(29) HPGPC detection results show that, the polysaccharide composition containing main FGAG peak of HSG-1, HSG-2 and HSG-3 has a weight average molecular weight (Mw) of about 63.2 kD, 63.5 kD and 64.2 kD respectively, and a number average molecular weight (Mn) of about 56.5 kD, 59.7 kD and 58.9 kD respectively; a polydispersity index (PDI) of about 1.12, 1.06 and 1.09, respectively. The result show that FGAG composition (main peak) in the polysaccharides of HSG-1, HSG-2 and HSG-3 exhibit a good uniformity.

(30) (3) Monosaccharide Composition Analysis:

(31) For the detection of HSG-1, HSG-2 and HSG-3, Elson-Morgon method, m-hydroxyldiphenyl method and Cysteine phenol method (Zhang weijie, Biochemical Research Technology of Glycoconjugate 2Ed, Zhejiang University Press, 1999) were used to detect the content of acetyl galactosamine, glucuronic acid and fucose in the polysaccharide composition. The detection results of monosaccharide composition of HSG-1, HSG-2 HSG-3 are shown in Table 1. The results show that, in the three monosaccharide compositions, the molar ratio of hexuronic acid, hexosamine and fucose is in the range of 1: (10.3):(10.5). In contrast, HSG-1 has higher fucose molar ratio, which is related to the peaks of the more residual FS polysaccharides (RT14 min), and HSG-2 and HSG-3 have relatively higher content of hexuronic acid and hexosamine, consistent with the effect of further purification.

(32) As shown in FIG. 1, HSG-2 basically do not have peak at Rt14 min, but its molar content of Fuc is still slightly higher than that of GlcUA and GaINAc, even its molar ratio of Fuc to GlcUA is slightly higher than that of HSG-3, which is in agreement with the find mentioned later that the polysaccharides contain FS, the molecular weight distribution of which is similar to that of FGAG.

(33) TABLE-US-00001 TABLE 1 Monosaccharide composition analysis of the Fuc3S4S substituted glycosaminoglycan-containing polysaccharide component from Holothuria scabra Mass percentage (%) Molar ratio GlcUA GalNAc Fuc GlcUA:GalNAc:Fuc HSG-1 15.62 18.93 16.65 1.00:1.05:1.28 HSG-2 16.58 19.12 15.33 1.00:0.99:1.11 HSG-3 16.64 19.82 15.06 1.00:1.03:1.09

Example 2

Preparation of Fuc3S 4S Substituted Oligoglycosaminoglycans-Containing Mixture by Peroxide Depolymerization of Polysaccharide Composition from Holothuria scabra (dHSG-1)

(34) 2.1 Materials

(35) HSG-2:

(36) polysaccharide composition containing Fuc3S4S substituted glycosaminoglycans prepared by extraction and purification of dried Holothuria scabra in Example 1. Mw, 63.5 kD; PDI, 1.06.

(37) Reagents:

(38) NaCl, CH.sub.3COONa, NaOH, H.sub.2O.sub.2, Cu (CH.sub.3COO).sub.2 and other commercially available reagents were analytical grade reagents.

(39) 2.2 Preparation

(40) 100.3 mg of HSG-2 obtained in Example 1 was placed in a 10 mL reaction tube, dissolved in 3 mL of water, 87.2 mg of NaCl was added to a concentration of 0.5M, 204.1 mg of sodium acetate trihydrate was added to a concentration of 0.5 M, 2.4 mg of copper acetate was added to a concentration of 4.01 nM, and 0.4 mL of 10% H.sub.2O.sub.2 was added. The reaction solution was adjusted with 0.1 M NaOH to pH about 7.5, and depolymerized at 35 C. 10 L sample was taken at intervals of 40 min, and subjected to alcohol precipitation and water dissolution, and detected by HPGPC to confirm the end of the reaction. The reaction was terminated after depolymerization for 3 hr. To the reaction solution 10 mg of disodium ethylenediaminetetraacetate was added, and then 7.5 mL of anhydrous ethanol was added, incubated without shaking and then centrifuged at 3000 rpm for 15 min. The precipitate was dissolved in 3 mL of water, and purified by OH.sup. type DEAE anion exchange resin column combined with H.sup.+ type cation exchange resin column, the fractions containing acidic mucopolysaccharides were collected, and adjusted with 0.1M NaOH solution to neutral pH, dialyzed with 1 kD dialysis bag for 48 hr. The dialysis retentate was freeze-dried, 75 mg of Fuc3S4S substituted oligglycosaminolycan product 1 (dHSG-1) was got.

(41) 2.3 Detection

(42) Methods:

(43) Molecular weight and distribution were measured by HPGPC. The content of acetylgalactosamine (D-GalNAc) was measured by Elson-Morgon method. The content of glucuronic acid (D-GlcUA) was measured by carbazole method. D-GalNAc/L-Fuc molar ratio was calculated by .sup.1H NMR methyl peak integral area. NMR spectra were got by AVANCE AV 500 superconducting nuclear magnetic resonance meter (500 MHz) (Bruker company, Switzerland).

(44) Results:

(45) The analysis results of physicochemical properties and monosaccharide compositions of HSG-2 and its peroxide depolymization product dHSG-1 are shown in table 1. Compared with dHSG-2, the Mw of dHSG-1 was significantly reduced by more than 7 fold. The monosaccharide composition analysis shows that, the composition ratio of hexosamine and hexuronic acid in HSG2 and dHSG-1 is basically stable, the ratio of deoxyhexose (Fuc) has been decreased, relate to the decrease of FS impurities.

(46) TABLE-US-00002 TABLE 2 Analysis results of physicochemical properties and monosaccharide compositions of HSG-2 and dHSG-1 from Holothuria scabra Molecular monosaccharide composition weight (molar ratio) Sample (Mw, kD) PDI GlcUA:GalNAc:Fuc HSG-2 63.5 1.06 1.00:0.99:1.11 dHSG-1 8.83 1.30 1.00:1.02:1.03

(47) Some spectra of NMR spectroscopy of dHSG-1 are shown in FIG. 2-3. .sup.1H NMR spectrum in FIG. 2A shows that between 5.0-5.7 ppm, there is a signal peak of -L-Fuc terminal at 5.32 ppm. Based on the correlation signals in .sup.1H-.sup.1H COSY, it can be seen that the proton signals at C2, C3, C4-positions are located at about 3.96, 4.53, 4.96 ppm, respectively. In further showed the C6-methyl signal is located at about 1.2-1.4 ppm, and the C5-proton signal is located at about 4.88 ppm. Compared with the corresponding proton signals of unsubstituted Fuc, the proton signals are shifted to downfield by more than 0.6 ppm, based on which it is concluded that sulfate substitution is present at C3, C4-positions. Obviously, Fuc contained in dHSG-1 is mainly -L-Fuc3S4S. It is concluded by combination of FIGS. 2 and 3 that, dHSG-1 contains a small amount of -L-Fuc4S and 2S4S. According to the integral proportion of -L-Fuc terminal hydrogen shown in .sup.1H NMR spectrum, it is concluded that in the fucose branches contained in dHSG-1, the proportion of Fuc3S4S, Fuc4S, Fuc2S4S are about 82%, 12% and 6% respectively.

(48) The .sup.13C NMR spectrum (FIG. 2B) shows that, terminal hydrogen signal of -L-Fuc3S4S is located at about 102 ppm, methyl signal is located at about 18.6 ppm, other C signals on its sugar ring can be clearly assigned by .sup.1H-.sup.13C HSQC correlation signals. Wherein compared with C signal at the corresponding position of unsubstituted Fuc, there are significant downfield shifts of C signal peaks at C3 and C4, indicating the presence of sulfate substitution at these positions.

(49) The terminal C signal of 13-D-GlcUA is located at about 105.6 ppm (FIG. 2B), HSQC spectrum shows that the terminal hydrogen signal is located at about 4.48 ppm, .sup.1H-.sup.1H COSY spectrum shows the proton signals at C2-C5 positions are located at about 3.62, 3.66, 3.94 and 3.70 ppm. Compared with the proton signal at the corresponding position of unsubstituted -D-GlcUA, proton signals at C3 and C2-positions are shifted downfield by about 0.3-0.5 ppm. It can be seen from the .sup.1H-.sup.1H ROESY signals (FIG. 3) that, -L-Fuc3S4S contained in HSG is linked to -D-GlcUA by 1-3 glycosidic linkage.

(50) C6 signal on -D-GlcUA is overlapped with the C7 signal on the -D-GalNAc, the other .sup.13C signals on sugar ring are assigned by .sup.1H-.sup.13C HSQC spectrum. C3 signal is located at about 82 ppm, which is shifted downfield by 3-4 ppm compared with the unsubstituted C3 signal, further demonstrating that there are substituted fucose branches at this position.

(51) By combination of the terminal .sup.13C signal (at about 102 ppm, FIG. 2B) of -D-GalNAc and .sup.1H-.sup.13C HSQC spectrum, it is concluded that the terminal proton signal is located at about 4.58 ppm. Based on the .sup.1H-.sup.1H COSY spectrum, it is concluded that C2-C5 proton signals are located at about 4.05, 3.92, 4.79 and 4.01 ppm, respectively, and C6 proton signal is located at about 4.23 and 4.31 ppm. Compared with the corresponding proton signals of unsubstituted -D-GalNAc, proton signals at C4, C6-positions are shifted downfield by about 0.5-0.7 ppm, thus it is concluded that the hexosamine contained in dHSG-1 is mainly -D-GalNAc4S6S. The characteristic methyl proton signal of acetyl on -D-GalNAc is located at about 2.04 ppm.

(52) TABLE-US-00003 TABLE 3 .sup.1H/.sup.13CNMR signal assignments of dHSG-1 H-1 H-2 H-3 H-4 H-5 H-6 H-8 -GalNAc-4S6S 4.58 4.05 3.92 4.79 4.01 4.23/4.31 2.05 -GlcUA 4.48 3.62 3.66 3.94 3.70 -Fuc-3S4S 5.32 3.96 4.53 4.96 4.88 1.36 C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 -GalNAc- 102.6 54.0 78.1 79.2 74.3 69.6 25.1 177.5 4S6S -GlcUA 105.8 75.9 81.8 78.0 79.0 177.5 -Fuc-3S4S 102.4 69.0 78.1 82.0 69.0 18.6

(53) Based on the .sup.13C NMR and DEPT135 spectra, methylene peaks at C6 position is located at about 69.6 ppm, which is shifted downfield by about 6 ppm compared with the C6 peak of unsubstituted -D-GlcUA, further demonstrating that sulfate substitution is present at C6. The characteristic signals of C2 and C8 on -D-GalNAc are located at about 54 ppm and about 25 ppm, which is consistent with the structure feature of acetylgalactosamine. The .sup.13C signals at C3-C5 can be assigned by .sup.1H-.sup.13C HSQC spectrum as shown in FIG. 3, and its C7 signal is overlapped with the C6 signal of -D-GlcUA.

(54) To summarize the chemical structure analysis, dHSG-1 is a kind of glycosaminoglycan derivative having -L-Fuc3S4S as main branch substitution (about 82%), with the basic structural unit is as follows:
{-4)-[L-Fuc3S4S-(1-]-3)-D-GlcUA-(1-3)-D-GalNAc4S6S-(-}.sub.n

(55) According to the above chemical structure analysis, it can be known that, with respect to the reported FGAG compositions, dHSG-1 has the characteristics as follows: (1) fucose branches are mainly -L-Fuc3S4S, and (2) there is 4,6-disulfated on GalNAc backbone. Compared with FGAG from Stichopus japonicas, Thelenota ananas and Brazilian sea cucumber, the branch substitution type of dHSG-1 is simple (mainly -L-Fuc3S4S), the C6 of backbone hexosamine is completely substituted by sulfated group (mainly -D-GalNAc4S6S), and therefore its chemical structure has better uniformity.

Example 3

Preparation of Fuc3S4S Substituted Oligoglycosaminoglycan Mixture by Deacylated Deaminated Depolymerization of the Polysaccharide Composition from Holothuria scabra (dHSG-2)

(56) 3.1 Materials

(57) HSG-3:

(58) polysaccharide composition containing Fuc3S4S substituted glycosaminoglycan obtained by extraction and purification of dried Holothuria scabra. Mw, 64.2 kD; PDI, 1.09.

(59) Reagents:

(60) hydrazine hydrate, hydrazine sulfate, anhydrous ethanol, sodium borohydride and other reagents were commercially available analytical reagents.

(61) 3.2 Preparation

(62) (1) Preparation of Partial Deacetylated Product:

(63) 360 mg of the polysaccharide composition HSG containing Fuc3S4S substituted glycosaminoglycan from Holothuria scabra in Example 1 was put in a reaction tube, added 14.5 mg of hydrazine sulfate as catalyst, and then added 1.45 mL of hydrazine hydrate, under the protection of nitrogen, stirred at 250 rpm, reacted at 70 C. for 12 hr. After the reaction completed, the reaction solution was added with ethanol to a concentration of ethanol of 80% (v/v), and centrifuged at 3000 rpm for 15 min. The precipitate was washed with 5 ml 80% (v/v) ethanol aqueous solution, dissolved in deionized water and dialyzed with a 3.5 kD dialysis bag in pure water for 3 days. Dialyzed retentate was freeze-dried to obtain 53.0 mg of partial deacetylated product of HSG-3. .sup.1H NMR detection showed that the deacetylation degree of the obtained product was about 12%.

(64) (2) Preparation of Deaminated Depolymerization Product dHSG-2:

(65) 40 mg of partial deacetylated product of HSG-3 of step 1 was accurately weighed and put into a reaction flask, and dissolved in 2 mL of water. Under the ice bath condition, 4 mL 5.5 M nitrous acid solution (pH 4) was added, depolymerized under ice bath for 10 min, and then 0.5 M NaOH solution was added to adjust pH to 9-10 to terminate the reaction.

(66) (3) Terminal Reduction and Product Purification:

(67) The reaction solution of step (2) was added with 2 mL of 0.1 mol/L sodium hydroxide solution containing 0.5 mol/L of sodium borohydride, heated at 50 C. for 40 min to reduce the aldehyde groups of nitrous acid depolymerization products. After the reaction completed, the reaction solution was cooled to room temperature, and 0.5 M H.sub.2SO.sub.4 was added dropwise to remove excessive sodium borohydride, and finally neutralized with 0.5 M NaOH, dialyzed with a 1.0 kDa dialysis bag in pure water for 24 hr. The resulting dialysis retentate was subjected to G50 column chromatography and with formetachromasia detection. The eluted fractions containing acidic mucopolysaccharide were collected and combined, and the resulting eluted solution was freeze-dried, about 38.5 mg of deaminated depolymerization product dHSG-2 was got.

(68) 3.3 Detection

(69) The detection method was the same as in Example 2. HPGPC monitoring results show that, after deacylated deaminated depolymerization, the resulting product still contains small amounts of polysaccharides with a Mw of about 60 kD (including FS and neutral glucan), the high molecular weight composition can be easily removed by G50 gel chromatography. The HSG-3 deaminated depolymerization product dHSG-2, which was purified by G50 gel chromatography, has a Mw of about 7.83 kD, and a polydispersity index (PDI) of about 1.38.

(70) The monosaccharide composition of dHSG-2 includes GlcUA, GalNAc and Fuc. The ratio of the three monosaccharide compositions was about 1.00:0.89:1.02. In dHSG-2 product the GalNAc content was slightly less than that of dHSG-1, which was basically in agreement with the integral area ratio of methyl signal peak of acetyl on GalNAc at about 2.02 ppm to methyl signal peak of Fuc at about 1.23-1.36 ppm showed in .sup.1H NMR spectrum, and was also basically in agreement with the deacetylation degree of partial deacetylation process.

(71) The .sup.1H, .sup.13C NMR and 2D correlation spectra show that, the main .sup.1H, .sup.13C signal peaks of dHSG-2 and the main correlation peaks in its 2D correlation spectrum are the same or similar to that of dHSG-1. However, dHSG-2 exhibits carbon, hydrogen signals assigned to 2,5 dehydrated talose (AnTol) different from those of dHSG-1.

(72) Based on the spin coupling system shown in .sup.1H-.sup.1H COSY spectrum, hydrogen in AnTol saccharide ring can be easily assigned, the two H signals at C1 are located at about 3.68 (H1) and 3.80 ppm (H1), the H signals at C2 to C5 are located at about 4.13 (H2), 4.54 (H3), 5.04 (H4) and 4.51 ppm (H5), respectively, and the two H signals at C6 are located at about 4.35 (H6) and 4.18 ppm (H6) respectively. Based on the heteronuclear correlation signals shown in .sup.1H-.sup.13C HSQC spectrum, the carbon signals of AnTol can be easily assigned, the signals of C1 to C6 are located at about 63.8, 83.9, 78.9, 80.4, 80.3 and about 71.0 ppm, respectively.

(73) Compared with the hydrogen and carbon signals at corresponding positions of unsubstituted AnTol, C4 and C6 signals of terminal AnTol of dHSG-2 are significantly shifted downfield, therefore, it is concluded that there is 4,6-disulfated substitution, i.e., the terminal AnTol group is AnTol4S6S. This is consistent with the structure of -D-GalNAc4S6S on backbone of dHSG-1, meanwhile it indicates that the sulfate groups are basically not affected during the deacylated deamination reaction.

(74) According to the integral proportion of terminal hydrogen of -L-Fuc in .sup.1H NMR spectrum, it is concluded that the fucose branches contained in dHSG-2 also includes Fuc3S4S, Fuc4S, Fuc2S4S, in which the proportion of Fuc3S4S is about 83%.

(75) In addition, it is demonstrated by the NMR spectrum of deacylated deaminated depolymerization product containing non-reduced terminal (2,5-dehydrated talose, AnTal) prepared in parallel by the present invention, that the C1 carbonyl of the terminal AnTal is easily bond to a molecule of water to form a -diol hydrate structure.

Example 4

Preparation of Fuc3S4S Substituted Oligoglycosaminoglycan Mixture (dHSG-3) by -Eliminative Depolymerization of Polysaccharide Composition from Holothuria scabra

(76) 4.1 Materials

(77) HSG-1:

(78) polysaccharide compositions containing Fuc3S4S substituted glycosaminoglycan which was extracted and purified from dried Holothuria scabra in Example 1. Mw, 63.2 kD; PDI, 1.09

(79) Reagents:

(80) benzethonium chloride, benzyl chloride, anhydrous ethanol, sulfuric acid, sodium hydroxide, metallic sodium and other reagents were commercially available analytical reagents.

(81) 4.2 Preparation

(82) (1) Preparation of quaternary ammonium salt of HSG

(83) 200.4 mg of HSG-1 was dissolved in 8 mL of deionized water. 500 mg of benzethonium chloride was dissolved in another 8 mL of deionized water. The benzethonium chloride solution was added dropwise into the HSG solution under the conditions of continuous shaking. White precipitate appeared in the solution. The solution was centrifuged at 4000 rpm for 20 min to obtain the precipitate, which was washed with 100 mL water for three times under shaking. The precipitate was dried in a vacuum oven under reduced pressure at room temperature for 24 hr and 490 mg of quaternary ammonium salt of HSG-1 was obtained.

(84) (2) Preparation of Partial Benzyl-Esterified Product of Carboxyl of Quaternary Ammonium salt of HSG-1

(85) 490 mg of quaternary ammonium salt of HSG-1 obtained in step 1 was put in a 50 mL round bottom flask, 13.2 mL of dimethylformamide (DMF) was added. After the sample was dissolved, 6.6 mL of benzyl chloride was added and reacted at 35 C. in a closed atmosphere for 2 hr with stirring at 500 rpm/min. After the reaction completed, 20 mL saturated NaCl and 160 mL anhydrous ethanol were added. The reaction solution was centrifuged at 4000 rpm for 15 min, and the precipitate was washed with 80% ethanol saturated with sodium chloride for three times. The resulting precipitate was dissolved in 10 mL of deionized water, and dialyzed using a dialysis bag with the molecular weight cutoff of 3.5 kD (dialyzed against running water for 12 hr, and against deionized water for 36 hr). Dialysis retentate was concentrated under reduced pressure to 5 mL, and a small amount of which was freeze-dried for .sup.1HNMR spectrum analysis. The results of .sup.1HNMR spectrum showed that the degree of carboxyl esterification was 26.9%. The rest of concentrated dialysis solution was subjected to cation exchange resin (H.sup.+ type, column bed 45 cm5 cm) to convert into its hydrogen type, and the eluted fractions containing acidic mucopolysaccharide were collected. Under the monitor of conductivity meter, the eluted fractions were titrated with 0.4 M tetrabutyl ammonium hydroxide to pH 8, and then the resulting solution was freeze dried, 245.5 mg of TBA type quaternary ammonium salt of partial benzyl-esterified product of HSG-1 was got.

(86) (3) -Eliminative Depolymerization

(87) 245.5 mg of TBA type quaternary ammonium salt of benzyl-esterified product of carboxyl obtained in step 2 was put into a 50 mL round-bottomed flask, added 4.9 mL DMF and freshly prepared 4.9 mL 0.04M EtONa/EtOH. Under 25 C. and nitrogen protection, reacted for 4 hr with stirring at 500 r/min. After the reaction completed, the reaction solution was sequentially added with 9.8 mL saturated NaCl solution and anhydrous ethanol to 100 mL, and then centrifuged at 4000 rpm for 15 min. The resultant precipitate was -eliminative depolymerization product of partial benzyl-esterified HSG-1(dHSG-3-0).

(88) 4) Debenzylation

(89) 10 mL 0.1 M NaOH was added into the precipitate dHSG-3-0 obtained in step 3 to dissolve it completely. After alkaline hydrolysis at 35 C. for 1 hr, 1 M HCl solution was added to adjusted pH to neutral. Anhydrous ethanol was added to a concentration of about 80% (v/v), and centrifuged at 4000 rpm for 15 min. The precipitate was dissolved in pure water, ultrafiltrated using a ultrafiltration tube having molecular weight cutoff of 30 kDa (study showed that such ultrafiltration step may effectively remove polysaccharide compositions that cannot be removed by -eliminative depolymerization, including fucan FS and neutral polysaccharide NP, which have molecular weight distribution close to or overlapped prototype FGAG from Holothuria scabra). The resulting filtrate with a molecular weight of less than 30 kDa was transferred into a 1 kD dialysis bag, and dialyzed against deionized water for 36 hr. The resulting dialysis retentate was freeze-dried, about 114 mg of Fuc3S4S substituted oligolycosaminoglycan mixture product (dHSG-3) was got.

(90) 4.3 Detection

(91) Detection of physicochemical properties and spectral analysis of dHSG-3 were the same as in Example 2.

(92) Analysis results of physicochemical properties and monosaccharide compositions of HSG-1 and its depolymerization product dHSG-3 are shown in Table 4. The detection results show that compared with HSG-1, the molecular weight of dHSG-3 is significantly decreased. The analysis results of monosaccharide composition show that compared with HSG-1, based on the molar ratio to GlcUA the proportion of GalNAc is increased, whereas the proportion of Fuc is decreased, the former is related to the -eliminative reaction of partial GlcUA, the latter is related to further removal of FS impurity in polysaccharide composition of HSG-1, and the molar ratio of monosaccharide composition GalNAc to Fuc contained in dHSG-3 is in agreement with the integral ratio of methyl signal peaks contained in such two glycosyl shown in .sup.1H NMR.

(93) TABLE-US-00004 TABLE 4 Detection results of physicochemical properties and monosaccharide compositions of HSG-1 and dHSG-3 from Holothuria scabra Molecular Optical Monosaccharide composition weight rotation (molar ratio) Sample (Mw, kD) PDI ([].sub.D.sup.20) GlcUA:GalNAc:Fuc HSG-1 63.2 1.26 68.5 1.00:1.05:1.28 dHSG-3 8.63 1.41 54.6 1.00:1.13:1.12

(94) A UV spectrophotometer was used to scan the sample with a wavelength range of 190 nm-400 nm. dHSG-3 has the maximum UV absorption at 232 nm, which is consistent with the presence of unsaturated bond UA.

(95) NMR spectrum results show that, compared with the spectrum of compound dHSG-1, the .sup.1H NMR spectrum of dHSG-3 has new signal peaks at about 5.76 and 5.82 ppm, according to the .sup.1H NMR correlation spectrum, these signals can be assigned to the characteristic signal of 4H on 4-deoxy-threo-hex-4-enopyranosyluronic acid-1-yl (UA), which is the -eliminative product of -D-GlcUA (Table 5, FIG. 4).

(96) The .sup.1H-.sup.1HCOSY spectrum and TOCSY spectrum of dHSG-3 clearly show that the signals of H4, H3, H2 and H1 of UA are coupling associated (FIG. 5). The ROESY spectrum shows that, L-Fuc branch is linked to -D-GlcUA or UA by (1-3) glycosidic linkage (FIG. 5). Compared with the terminal H signal of L-Fuc linked to -D-GlcUA, the terminal H signal of Fuc linked to UA is obviously present at lower field (FIG. 4).

(97) In the .sup.13C-NMR spectrum, C1 peaks of -D-GlcUA and -D-GalNAc are present at about 97-104 ppm, while C1 peak of UA is present at about 103.5 ppm, and the chemical shift of C4 is at 106.8 ppm, the chemical shift of C5 is at about 148.5 ppm (FIG. 4).

(98) TABLE-US-00005 TABLE 5 .sup.1H/.sup.13C NMR signal assignment of compound dHSG-3 ( [ppm]) H-1 H-2 H-3 H-4 H-5 H-6 H-8 -GalNAc4S6S 4.56 4.05 3.99 4.75 4.03 4.25, 4.33 2.04 4.59 4.11 4.16 4.98 4.08 4.27, 4.37 2.09 UA 4.49 3.62 3.67 4.01 3.70 -GlcUA 4.91 3.88 4.66 5.74 -Fuc3S4S 5.32 3.95 4.53 4.97 4.82 1.38 5.25 3.96 4.59 4.88 4.32 1.28 C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 -GalNAc4S6S 102.2 54.3 78.1 78.8 74.3 69.6 25.1 177.5 102.2 54.1 78.1 78.8 74.3 69.6 25.1 177.5 UA 105.8 75.8 81.5 78.0 79.6 169.0 -GlcUA 103.5 72.6 80.3 109.2 177.5 -Fuc3S4S 101.9 68.0 78.0 81.8 68.0 18.6 100.5 68.0 78.0 81.8 68.0 18.6

(99) Taking the hydrogen spectrum, carbon spectrum and correlation spectra into consideration, in the main chain monosaccharide compositions of dHSG-3, D-GlcUA and D-GalNAc are linked to each other by (1.fwdarw.3) and (1.fwdarw.4) glycoside linkages to constitute a polysaccharide backbone, and thus form a disaccharide structure unit. According to the chemical shifts of H2, H3 of GlcUA combined with .sup.1H-.sup.1H ROESY and .sup.1H-.sup.13C HMBC, it is concluded that L-Fuc is linked to -D-GlcUA by (1.fwdarw.3) glycosidic linkage, and the main L-Fuc branch type is Fuc3S4S. Obviously, in dHSG-3, non-reducing terminal hexuronic acid is mainly UA.

(100) It is concluded based on the -L-Fuc terminal hydrogen integral ratio of -L-Fuc shown in the .sup.1H NMR spectrum, fucose branch contained in dHSG-3 includes the structure types, such as -L-Fuc3S4S, -L-Fuc4S, -L-Fuc2S4S, and the main type is -L-Fuc3S4S. Based on molar ratio, -L-Fuc3S4S accounts for about 82% of the total fucosyl residues.

Example 5

Carboxyl Reduction and Reductive Amination of Reducing Terminal Carbonyl of Fuc3S4S Substituted Oligoglycosaminogly Mixture (dHSG-3)

(101) 5.1 Materials

(102) dHSG-3:

(103) Fuc3S4S substituted oligoglycosaminogly mixture obtained in Example 4.

(104) Reagents:

(105) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-phenyl-3-methyl-5-pyrazolone (PMP), tyramine hydrochloride, sodium cyanoborohydride, sodium borohydride, HCl, NaCl and other reagents were commercially available analytical reagents.

(106) 5.2 Preparation

(107) (1) Preparation of Carboxyl Reductive Derivative:

(108) 30 mg dHSG-3 was dissolved in 5 ml water, and adjusted to pH 4.75 with 0.1 N HCl. 90 mg EDC was added within 5 mins, and adjusted with 0.1 N HCl to maintain pH at 4.75. 360 mg NaBH.sub.4 was added slowly with stirring, and the reaction solution was put in water bath at 50 C. for 2 hr. A small amount of acetic acid was added dropwise to remove the excessive NaBH.sub.4, and then dialyzed against deionized water with a 3.5 KD dialysis bag. The dialysis retentate was freeze-dried about 24.6 mg of carboxyl reductive Fuc3S4S substituted oligoglycosaminogly mixture was got (Yield 78.3%).

(109) (2) Preparation of Terminal Reductive Aminated Derivative (2):

(110) 30 mg dHSG-3 was dissolved in 2.5 ml 0.2 mM phosphate buffer (PBS, pH 8.0) and with stirring sequentially added 24 mg tyramine hydrochloride and 9 mg sodium cyanoborohydride, incubated in water bath at 35 C. for 5 d, added 7.5 ml of 95% ethanol, centrifuged at 3000 rpm for 15 min. The precipitate was washed with 3 ml 95% ethanol twice, evaporated under reduced pressure to remove ethanol. The washed precipitate was dissolved in 2 ml of 0.1% NaCl, centrifuged at 3000 rpm for 15 min to remove insoluble matters. The supernatant was transferred into a 3.5 kD dialysis bag, dialyzed against deionized water (100 ml3) for 36 hr, and the dialysis retentate was freeze-dried, about 20.5 mg terminal reductive aminated Fuc3S4S substituted oligoglycosaminogly mixture was got (Yield 68%).

(111) 5.3 Detection

(112) (1) dHSG-3a Detection:

(113) IR spectroscopy of dHSG-3a was measured using a liquid pool detection method according to the reference (Carbohydr. Res, 1978, 63: 13-27); The molar ratio of OSO.sub.3.sup./COO.sup. was determined by conductance titration method (Weijie Zhang, Biochemical Research Technology of Glycoconjugate 2Ed, Zhejiang University Press, 1999, 409-410). The results show that there is no signal of carboxyl of -D-GlcUA in the liquid pool IR spectrum of dHSG-3a, suggesting that -D-GlcUA has been reduced into -D-Glc; the results of conductivity detection show that there is only one inflection point in the conductivity titration curve, the inflection point is from the sulfate group. No inflection point on the curve of conductivity change due to carboxyl group indicates that dHSG-3 in the -D-GlcUA has been reduced.

(114) (2) dHSG-3b Detection:

(115) Monosaccharide composition and NMR spectrum of dHSG-3b were analyzed according to the method as the same in Example 1. The results show that in the monosaccharide composition of dHSG-3b, the molar ratio of D-GalNAc:D-GlcUA:L-Fuc is about 1.00:1.02:1.11, which is basically in agreement with the theoretical calculation result of structural unit of dHSG-3b: .sup.1HNMR (D.sub.2O, [ppm]): 7.25 (2, 6H); 6.91 (3, 5H); 5.74 (UA, 4H), 5.32, 5.25 (L-Fuc1H); 3.38 (8H); 2.82 (7H); 2.02 (D-GalNAc, CH3); 1.26-1.36 (L-Fuc, CH.sub.3). Integral ratio of methyl hydrogen to benzene hydrogen of L-Fuc is about 11, indicating that the reducing terminal of the resulting product is all reductively tyraminated.

Example 6

Comparison of Anticoagulant Activities of Native FGAG and Depolymerization Product with Different Sulfation Patterns of Fuc Branches

(116) 6.1 Materials

(117) (1) FGAGs:

(118) AJG, TAG, IBG, LGG, HSG and PGG, prepared by extraction of commercially available dried Apostichopus japonicus, Thelenota ananas, Isostichopus badionotus, Ludwigothurea grisea, Holothuria scabra and Pearsonothuria graeffei, based on the preparation method of HSG-2 in Example 1.

(119) (2) dFGAGs:

(120) dAJG, dTAG, dIBG, dLGG, dHSG and dPGG, prepared by peroxide depolymerization and purification using AJG, TAG, IBG, LGG, HSG and PGG as starting materials, similar to the preparation method of dHSG-1 in Example 2.

(121) (3) Reagents and Instruments:

(122) Human quality control plasma, activated partial thromboplastin time (APTT) kit, prothrombin time (PT) kit were from Teco Medical (Germany); low molecular weight heparin (LMWH), enoxaparin sodium were from Sanafi Aventis company (France); prekallikrein (PK), kallikrein substrate, thrombin (IIa) 100 NIHU/mg, thrombin chromogenic substrate (CS-0138) 25 mg/vial, heparin cofactor II (HC-II) 100 g/vial, f.VIII detection kit were from HYPHEN BioMed company (France); factor VIII (f.VIII) 200 IU/single were from Shanghai RAAS blood products company; dermatan sulfate (DS) was from Sigma company (United States), oversulfated hondrointin sulfate (OSCS) was from Serva Electro-phoresis GmbH company (Germany); microplate reader was from Bio Tek Inc. (US); Chronolog-700 platelet aggregation meter (US).

(123) 6.2 Methods

(124) (1) Detection of Anticoagulant Activity:

(125) The FGAGs, dFGAGs and reference samples were prepared into a series of concentrations using saline solution. Quality control plasma, detection kit and MC-4000 coagulation analyzer were used to detect the effect of the sample solutions on APTT and PT of human quality control plasma according to the instructions of the kits.

(126) (2) Detection of Inhibition Activity on Endogenous Factor X Enzyme (f.Xase):

(127) 30 L of each of series concentration of FGAG and dFGAG solutions and 30 L of f.VIII (2 IU/mL) were mixed, and according to the method of the instruction of the f.VIII detection kit, f. IXa reagent, f.Xa reagent and f.Xa substrate (SXa-11) were sequentially added. OD.sub.405 nm was measured. EC.sub.50 values of f.Xase inhibition by each sample were calculated according to the method of reference (Blood, 2006, 107:3876-3882).

(128) (3) Test of HCII-Dependent Antithrombin (f.IIa) Activity:

(129) The sample solutions at a series of concentrations were added into 96-well plates, respectively, and then sequentially added 30 L HC-II (1 M), f.IIa (10 U/mL) and CS-0138 (4.5 mM, chromogenic substrate), incubated at 37 C. for 2 min. OD.sub.405 nm was measured and EC.sub.50 values of IIa inhibition by each sample were calculated.

(130) (4) Detection of F.XII Activation:

(131) 30 L sample and reference solution at a series of concentrations were added into 96-well plates, respectively, and then sequentially added 30 L f.XII (25 g/mL), 30 L PK (620 nM) and 30 L chromogenic substrate, incubated at 37 C. for 1 min. OD405 nm was measured.

(132) (5) Detection of Platelet Activation Activity:

(133) Anticoagulant platelet-rich plasma (PRP) and platelet poor plasma (PPP) were collected from healthy volunteers. The platelet aggregation-inducing activity of the FGAG, dFGAG sample solutions at a series of concentrations was measured by turbidimetry using Chronolog-700 platelet aggregation meter, wherein the FGAG, dFGAG sample solutions were prepared by dissolving FGAG, dFGAG in saline solution.

(134) 6.3 Results

(135) (1) Anticoagulant Activity:

(136) The anticoagulant activities of FGAGs, dFGAGs and reference are shown in Table 6.

(137) Different sulfation patterns and their composition ratios of fucose branches of FGAGs and dFGAGs from different species were calculated according to the integral of .sup.1H NMR spectra of dFGAGs (FIG. 6). It can be seen from the data shown in Table 6 that, the concentration for doubling APTT of human quality control plasma of native FGAGs is at about 2.2-3.3 g/ml. The difference among the effect on APTT activity between different FGAGs is not significant; the difference among the activities of dFGAGs after depolymerization is increased, wherein the dIBG containing Fuc2S4S as main branches has the strongest activity, and dHSG is close to it, dLGG has the weakest activity.

(138) Some FGAGs have IC.sub.50 values of f.Xase inhibition of between about 12.2-19.4 ng/ml, and the difference among their activity potency is not significant. After depolymerization, except that the activities of dAJG and dLGG are weaker than that of the native forms, the activities of other dFGAGs are stronger than that of the native forms, whereas the differences of the potency of f.Xase inhibition activities among dFGAGs are increased, wherein dIBG has the strongest activity, and dHSG had relatively strong activity, dLGG has the weakest activity.

(139) Among the several FGAGs, the HCII-dependent anti-f.IIa activities are within the range of about 300-400 ng/ml. After depolymerization, the activities of the dFGAGs are all stronger than that of the native forms, IC.sub.50 values are within the range of about 130-250 ng/ml. In relative terms, HSG and dHSG have the strongest activities, whereas IBG and dIBG have the weakest activities.

(140) Obviously, PGG/dPGG has the L-Fuc sulfation features similar to HSG/dHSG. Correspondingly, the potency of its anticoagulant activity and the activity of coagulation factor activity inhibition are also basically the same.

(141) TABLE-US-00006 TABLE 6 Anticoagulant activities of FGAGs and their depolymerization product dFGAGs Sulfation pattern of Fuc branch Molecular (%) (%).sup.a SO.sub.4.sup./ 2 APTT.sup.b Xase.sup.c IIa/HCII.sup.c weight (kD) 2S4S 3S 4S 3S4S 0S COO.sup.a (g/ml) (ng/ml) (ng/ml) AJG 63 60 / 10 30 / ~3.9 2.20 12.2 375 dAJG 12.5 5.64 16.8 206 TAG 66 59 9 14 18 / ~3.6 2.49 14.2 409 dTAG 11.6 5.78 10.6 144 IBG 65 92 / 4 4 / ~4.0 2.50 14.4 497 dIBG 13.8 3.48 9.9 248 LGG 60 10 30 22 21 17 ~2.5 3.36 18.6 381 dLGG 10.3 8.35 22.2 253 HSG 62 5 / 10 85 / ~3.9 2.97 19.4 297 dHSG 10.2 3.73 12.4 132 PGG 60 2 / 16 82 / ~3.8 2.86 18.2 302 dPGG 12.2 3.80 11.8 137 LMWH 4.5~5.5 / >1.8 7.80 68.8 184 DS 42 / ~1.4 65.1 2549 67 .sup.aMole percentage or molar ratio; .sup.bdrug concentration required for doubling APTT; .sup.cbased on EC.sub.50;

(142) (2) Detection of f.XII Activation Activity:

(143) The activation activity of FGAGs from different species and their depolymerization product dFGAGs on f.XII are shown in FIG. 7. Compared with other FGAGs, the f.XII activation activities of HSG and PGG with Fuc3S4S as main fucose branch are significantly weak, and their corresponding depolymerization products also have the similar feature. In contrast, the content of sulfate groups on the backbone and branches of HSG are higher than that of LGG, but slightly higher than that of TAG, close to that of AJG, and slightly lower than that of IBG, whereas their f.XII activation activities are all significantly lower, indicating that f.XII activation of FGAG compositions is not just dependent on the loading capacity of its negative charge, but is related to sulfation patterns of the branch.

(144) (3) Detection of Platelet Activation Activity:

(145) Effect of FGAGs and dFGAGs on platelet activity is shown in Table 7.

(146) TABLE-US-00007 TABLE 7 Effect of FGAGs and their depolymerization product dFGAGs on platelet aggregation in healthy volunteers (n = 3) Aggregation Aggregation Sample rate (%) Sample rate (%) Con. 9 4 ADP 10 M 93 7 AJG 30 g/ml 62 15 dAJG 60 g/ml 27 11 TAG 30 g/ml 28 6 dTAG 60 g/ml 14 3 IBG 30 g/ml 65 20 dIBG 60 g/ml 76 14 LGG 30 g/ml 27 11 dLGG 60 g/ml 35 8 HSG 30 g/ml 13 6 dHSG 60 g/ml 9 3 PGG 30 g/ml 14 5 dPGG 60 g/ml 8 3

(147) It can be seen from the results shown in Table 7, HSG and PGG with Fuc3S4S as main fucose branch also have significantly weaker platelet activation activity in healthy volunteers, and their corresponding depolymerization products have the similar feature. Compared with the other dFGAGs, dIBG has the strongest platelet activation activity, while dLGG having a lower negative charge loading capacity still has certain platelet activation activity. The results show that the platelet activation activities of FGAG and dFGAG are also associated with the sulfation patterns of the fucose branches.

(148) The experimental results of this example show that the FGAG compositions with Fuc3S4S as main fucose branch have more advantageous anticoagulant antithrombotic activity, and have potent f.Xase inhibition activity and potent anticoagulant activity of HCII-dependent anti-f.IIa activity, but have no or less f.XII and platelet activation activity.

Example 7

Comparison of Anticoagulant Activities of dHSG Products Obtained by Different Depolymerization Methods

(149) 7.1 Materials

(150) (1) Fuc3S4S Substituted Oligoglycosaminoglycan Mixture Samples:

(151) HSG-2 (Mw 63.5 kD, PDI1.06), the sample obtained in Example 2;

(152) dHSG-4 (Mw 10.9 kD, PDI1.35), prepared by peroxide depolymerization;

(153) dHSG-5 (Mw 7.5 kD, PDI1.28), prepared by deaminated depolymerization (terminal reduction);

(154) dHSG-6 (Mw 8.4 kD, PDI1.33), -eliminative depolymerization (terminal tyramine reductive amination)

(155) (2) Reagents and Instruments:

(156) The same as in Example 6.

(157) 7.2 Methods:

(158) The same as in Example 6.

(159) 7.3 Results:

(160) The anticoagulant activities of dHSG-4-6 are shown in Table 8.

(161) TABLE-US-00008 TABLE 8 Anticoagulant activities of HSG and its depolymerization products Anticoagulant activity Effect on clotting factor activity 2 APTT.sup.a PT.sup.b TT.sup.b f.Xase.sup.a f.IIa/HCII.sup.a Sample (g/mL) (s) (s) (ng/ml) (ng/ml) HSG-2 2.97 15.6 24.7 21.5 334 dHSG-4 4.73 14.2 14.1 12.6 137 dHSG-5 4.95 13.7 14.0 15.3 113 dHSG-6 4.97 14.5 14.2 13.5 107 .sup.athe same as noted in Table 6; .sup.bCoagulation time at a drug concentration of 16 g/mL (PT, TT values in control group are 14.0 s and 13.6 s);

(162) The results show that the HSG depolymerization products obtained by different methods have potent anticoagulant, f.Xase inhibition and HCII-dependent antithrombin activity. Wherein the anticoagulant activity is mainly expressed as the inhibition of endogenous coagulation (significant extension of APTT), and there is no effect on exogenous coagulation (no extension of PT). Prototype HSG-2 has a larger effect on common coagulation pathway (extension of TT, HSG-2 has an ATIII-dependent antithrombin activity), and the depolymerization samples have weaker effect on TT.

Example 8

Anticoagulant Activities of dHSGs with Different Molecular Weights

(163) 8.1 Materials

(164) (1) Samples:

(165) Similar to the method for the preparation of HSG-2 in Example 1. HSG-a with Mw 63.6 kD was extracted and purified from commercially available dried Holothuria scabra, and further purified by ion exchange column chromatography and converted into Na.sup.+ salt.

(166) dHSG-1a (Mw 49.0 kD), dHSG-2a (Mw 27.8 kD), dHSG-3a (Mw 14.9 kD), dHSG-4a (Mw 8.24 kD), dHSG-5a (Mw 5.30 kD), dHSG-6a (Mw 3.12 kD) were all depolymerization products of HSG-a, prepared according to the depolymerization method similar to -eliminative depolymerization in Example 1, and the reductive terminals were all reduced into alditol groups.

(167) (2) Reagents and Instruments:

(168) The same as in example 6.

(169) 8.2 Methods:

(170) Same as in Example 6.

(171) 8.3 Results:

(172) Anticoagulant activities of dHSG-1a-6a are shown in Table 9 and FIG. 8.

(173) TABLE-US-00009 TABLE 9 Anticoagulant activities of HSG and its depolymerization products 2 APTT.sup.a f.Xase.sup.a HCII/IIa.sup.a Sample Mw (g/mL) (ng/mL) (ng/mL) HSG-a 63.6 2.62 25.9 589.2 dHSG-1a 49.0 2.32 17.9 542.9 dHSG-2a 27.8 2.18 16.2 455.9 dHSG-3a 14.9 3.16 15.8 315.9 dHSG-4a 8.24 3.09 15.6 220.7 dHSG-5a 5.30 4.36 23.4 151.5 dHSG-6a 3.12 9.90 79.5 285.5 LWMH 6.55 81.8 230.8 .sup.athe same as noted in Table 6;

(174) It can be seen from table 9 that, calculated based on mass concentration both the f.Xase inhibition activity and the HCII-dependent antithrombin activity of the depolymerization products with Mw in the range from 3 kD to 60 kD show a phenomenon of gradual increase and then decrease with the decrease of the molecular weight. The f.Xase inhibition activity is the strongest when Mw is about 8 kD, and the HCII-dependent antithrombin activity is the strongest when Mw is about 5 kD.

(175) Previously it has been found from the research about the relationship between the anticoagulant activity of TAG depolymerization product and molecular weight that, Mw is less than about 1012 kD, the f.Xase inhibition activity of TAG depolymerization products is gradually weakened, whereas when Mw is about 8 kD, the HCII-dependent antithrombin activity is basically similar and thereafter gradually weakened. Obviously, it can be seen from the correlation between molecular weight and anticoagulant activity that, the depolymerization product of HSG with lower molecular weight has more significant advantageous characteristics.

(176) Considering the physicochemical property, pharmacological activity and safety of the polysaccharide compounds, the suitable weight-average molecular weights of HSG as well as the structure similar FGAG depolymerization product with Fuc3S4S as main focus component in the branch are in the range of about 3 kD-10 kD, and a more suitable weight-average molecular weight may be selected in the range of about 4.5 kD-9 kD.

Example 9

Preparation of Freeze-Dried Powder for Injection of Low-Molecular-Weight Fucosylated Glycosaminoglycan

(177) 9.1 Materials

(178) dHSG:

(179) prepared according to the method similar to dHSG-3a described in Examples 4, with 5. Mw 5.5 kDa and PDI 1.20.

(180) 9.2 Formula

(181) TABLE-US-00010 Name of raw material and excipent Dosage dHSG 50 g Water for injection 500 mL Prepared into 1000
9.3 Preparation Process

(182) The formulated low-molecular-weight fucosylated glycosaminoglycan sodium salt was weighed and added with water for injection to full capacity, stirred to dissolve completely, and subjected to interval autoclaving sterilization. 0.6% pharmaceutical activated carbon was added and stirred for 20 min. A Buchner funnel and a 3.0 m micro porous filter membrane were used for decarbonization filtration to remove pyrogens. The content of the intermediate was tested. The qualified products were passed through a 0.22 m micro-porous filter membrane, filled into penicillin bottles with 0.5 mL in each bottle, partially stoppered, and transported into a lyophilizer and lyophilized according to the predetermined freeze-drying curve, completely stoppered, withdrawn from the lyophilizer, capped, inspected for qualification, and packed to obtain the final products.

(183) Lyophilization procedure: Put the samples into the lyophilizer, lowered the temperature of shelves to 40 C. and kept at 40 for 3 hr. Lowered the temperature of cold trap was to 50 C., and the vacuum was pumped to 300 bar. Sublimation: the temperature was increased at constant speed to 30 C. within 1 hr and maintained at 30 C. for 2 hr, Then the temperature was increased at constant speed to 20 C. within 2 hr and maintained at 20 C. for 8 hr, and the vacuum was maintained at 200-300 bar. Drying: The temperature was increased to 5 C. within 2 hr and maintained at 5 C. for 2 hr, and the vacuum was maintained at 150-200 bar. Then the temperature was increased to 10 C. within 0.5 hr, and maintained at 10 C. for 2 hr, and the vacuum was maintained at 80-100 bar. Then the temperature was increased to 40 C. within 0.5 hr and maintained at 40 C. for 4 hr, and the vacuum was reduced to the lowest.