SULFATED HEPARIN OLIGOSACCHARIDE AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Provided are a sulfated heparin oligosaccharide as well as a preparation method and an application thereof. The sulfated heparin oligosaccharide molecule contains an unsaturated double bond resulting from enzymolysis by heparinase at the non-reducing end thereof, an uronic acid derivative and a glycosylamine derivative; and has a structure represented by formula I, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.a, R.sub.b, R.sub.c, R.sub.d; R.sub.x, R.sub.y, R.sub.z, and n are as defined herein. The preparation method obtains a sulfated oligosaccharide with a controllable degree of sulfation. The sulfated heparin oligosaccharide has a high activity for inhibiting heparanase in vitro, with an activity 4-5 times higher than that of heparin for inhibiting cell adhesion and migration, and an activity 2-3 times higher than that of heparin for resisting tumor metastasis in mice, thus having a relatively good effect in resisting tumor metastasis and a relatively high specificity.

Claims

1. A sulfated heparin oligosaccharide, characterized in that the sulfated heparin oligosaccharide molecule contains an unsaturated double bond resulting from enzymolysis by heparinase at the non-reducing end thereof, comprises an uronic acid derivative and a glycosylamine derivative, and has a structure represented by Formula I: ##STR00004## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.a, R.sub.b, R.sub.c and R.sub.d are independently SO.sub.3.sup. or H; R.sub.x, R.sub.y and R.sub.z are independently COCH.sub.3 or SO.sub.3.sup., and n is 1 to 3.

2. The sulfated heparin oligosaccharide according to claim 1, characterized in that in Formula I, the number of sulfonic groups in each disaccharide unit is no less than 2 on average, the number of acetyl groups in each disaccharide unit is no more than 0.5 on average, and the numbers of sulfonic groups at position 6 and position 3 in glucosamine in each disaccharide unit are both no less than 0.5 on average; preferably, the uronic acid in Formula I is glucuronic acid or iduronic acid; and preferably, the cation forming a salt with the carboxyl and/or sulfonic group contained in Formula I is selected from the group consisting of Na.sup.+, K.sup.+ or Ca.sup.2+.

3. The sulfated heparin oligosaccharide according to claim 1, characterized in that the sulfated heparin oligosaccharide is any one of, or a combination of at least two of, the compounds having the following structures: DP6, a heparin hexasaccharide derivative: U.sub.3S-A.sub.NS3S-I-A.sub.NS3S-G.sub.3S-A.sub.NS3S6S U.sub.3S-A.sub.NS-I.sub.3S-A.sub.NS3S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-G.sub.2S3S-A.sub.NS3S6S DP8, a heparin octasaccharide derivative: U.sub.3S-A.sub.NS3S-I-A.sub.NS3S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.3S-A.sub.NS-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S DP10, a heparin decasaccharide derivative: U.sub.3S-A.sub.NS3S-I-A.sub.NS3S-I-A.sub.NS6S-I-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.3S-A.sub.NS-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS6S-I.sub.3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS6S-I.sub.2S3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-G.sub.2S3S-A.sub.NS3S6S.

4. A preparation method for the sulfated heparin oligosaccharide according to claim 1, characterized in that the method comprises the following steps: (1) degrading heparin with heparinase, separating and purifying to obtain a heparin oligosaccharide; and (2) sulfating the heparin oligosaccharide obtained in step (1) with a sulfating reagent to obtain the sulfated heparin oligosaccharide.

5. The preparation method according to claim 4, characterized in that the heparinase in step (1) is heparinase I.

6. The preparation method according to claim 4, characterized in that a buffer, preferably a Tris-HCl buffer, pH 7.0, needs to be added when heparin is degraded with heparinase in step (1); preferably, the amount of heparinase added in step (1) is 15 to 25 IU/g heparin, preferably 18 to 23 IU/g heparin; preferably, the temperature for the degradation of heparin with heparinase in step (1) is 4 C. to 37 C., preferably 8 C. to 25 C.; preferably, the time for the degradation in step (1) is 8 to 24 hours, preferably 10 to 20 hours; preferably, the degradation of heparin with heparinase in step (1) comprises inactivation at 95 C. for 5 to 10 min, preferably 5 to 8 min, and more preferably 6 min, after the degradation; preferably, the separation in step (1) comprises ultrafiltration, preferably ultrafiltration with a 10 KDa ultrafiltration centrifuge tube; and preferably, the purification in step (1) is separation and purification through column chromatography.

7. The preparation method according to claim 4, characterized in that before the sulfation in step (2), the heparin oligosaccharide obtained in step (1) is subjected to swelling treatment; and preferably, the solvent used for the swelling treatment is DMF.

8. The preparation method according to claim 4, characterized in that the sulfating reagent in step (2) is (CH.sub.3).sub.3N.SO.sub.3; preferably, relative to 1 g of the heparin oligosaccharide, the amount of the sulfating reagent is 1 to 10 g; preferably, the temperature for the sulfation in step (2) is 60 C. to 120 C.; and preferably, the time for the sulfation in step (2) is 1 to 12 hours.

9. The preparation method according to claim 4, characterized in that the method comprises the following steps: (1) degrading heparin with heparinase I, which is added at an amount of 15 to 25 IU/g heparin, at 4 C. to 37 C. for 8 h to 24 h in a buffer being a Tris-HCl buffer, pH 7.0, and after the degradation, inactivating at 95 C. for 5 to 10 min, ultrafiltering with a 10 KDa ultrafiltration centrifuge tube, and then separating and purifying through column chromatography to obtain the heparin oligosaccharide; and (2) sulfating the heparin oligosaccharide obtained in step (1) at 60 C. to 120 C. for 1 to 12 hours with a sulfating reagent to obtain the sulfated heparin oligosaccharide.

10. A method for anti-tumor metastasis, comprising administering an effective amount of the sulfated heparin oligosaccharide according to claim 1 to a subject in need thereof.

11. The sulfated heparin oligosaccharide according to claim 1, characterized in that the sulfation degree of the sulfated heparin oligosaccharide is from 40% to 60%.

12. The sulfated heparin oligosaccharide according to claim 2, characterized in that the sulfated heparin oligosaccharide is any one of, or a combination of at least two of, the compounds having the following structures: DP6, a heparin hexasaccharide derivative: U.sub.3S-A.sub.NS3S-I-A.sub.NS3S-G.sub.3S-A.sub.NS3S6S U.sub.3S-A.sub.NS-I.sub.3S-A.sub.NS3S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-G.sub.2S3S-A.sub.NS3S6S DP8, a heparin octasaccharide derivative: U.sub.3S-A.sub.NS3S-I-A.sub.NS3S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.3S-A.sub.NS-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S DP10, a heparin decasaccharide derivative: U.sub.3S-A.sub.NS3S-I-A.sub.NS3S-I-A.sub.NS6S-I-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.3S-A.sub.NS-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S-I.sub.3S-A.sub.NS6S-I.sub.3S-A.sub.NS6S-G.sub.3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS3S6S-I.sub.3S-A.sub.NS6S-I.sub.3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS6S-I.sub.2S3S-A.sub.NS6S-G.sub.2S3S-A.sub.NS3S6S U.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-I.sub.2S3S-A.sub.NS3S6S-G.sub.2S3S-A.sub.NS3S6S.

13. A preparation method for the sulfated heparin oligosaccharide according to claim 2, characterized in that the method comprises the following steps: (1) degrading heparin with heparinase, separating and purifying to obtain a heparin oligosaccharide; and (2) sulfating the heparin oligosaccharide obtained in step (1) with a sulfating reagent to obtain the sulfated heparin oligosaccharide.

14. The preparation method according to claim 13, characterized in that the heparinase in step (1) is heparinase I.

15. The preparation method according to claim 13, characterized in that a buffer, preferably a Tris-HCl buffer, pH 7.0, needs to be added when heparin is degraded with heparinase in step (1); preferably, the amount of heparinase added in step (1) is 15 to 25 IU/g heparin, preferably 18 to 23 IU/g heparin; preferably, the temperature for the degradation of heparin with heparinase in step (1) is 4 C. to 37 C., preferably 8 C. to 25 C.; preferably, the time for the degradation in step (1) is 8 to 24 hours, preferably 10 to 20 hours; preferably, the degradation of heparin with heparinase in step (1) comprises inactivation at 95 C. for 5 to 10 min, preferably 5 to 8 min, and more preferably 6 min, after the degradation; preferably, the separation in step (1) comprises ultrafiltration, preferably ultrafiltration with a 10 KDa ultrafiltration centrifuge tube; and preferably, the purification in step (1) is separation and purification through column chromatography.

16. The preparation method according to claim 13, characterized in that before the sulfation in step (2), the heparin oligosaccharide obtained in step (1) is subjected to swelling treatment; and preferably, the solvent used for the swelling treatment is DMF.

17. The preparation method according to claim 13, characterized in that the sulfating reagent in step (2) is (CH.sub.3).sub.3N.SO.sub.3; preferably, relative to 1 g of the heparin oligosaccharide, the amount of the sulfating reagent is 1 to 10 g; preferably, the temperature for the sulfation in step (2) is 60 C. to 120 C.; and preferably, the time for the sulfation in step (2) is 1 to 12 hours.

18. The preparation method according to claim 13, characterized in that the method comprises the following steps: (1) degrading heparin with heparinase I, which is added at an amount of 15 to 25 IU/g heparin, at 4 C. to 37 C. for 8 h to 24 h in a buffer being a Tris-HCl buffer, pH 7.0, and after the degradation, inactivating at 95 C. for 5 to 10 min, ultrafiltering with a 10 KDa ultrafiltration centrifuge tube, and then separating and purifying through column chromatography to obtain the heparin oligosaccharide; and (2) sulfating the heparin oligosaccharide obtained in step (1) at 60 C. to 120 C. for 1 to 12 hours with a sulfating reagent to obtain the sulfated heparin oligosaccharide.

19. A method for anti-tumor metastasis, comprising administering an effective amount of the sulfated heparin oligosaccharide according to claim 2 to a subject in need thereof.

20. The sulfated heparin oligosaccharide according to claim 2, characterized in that the sulfation degree of the sulfated heparin oligosaccharide is from 40% to 60%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 is a graph showing the results of separation of the heparin oligosaccharides by a Bio-Gel P-10 (2.5100 cm) chromatographic column in Example 1 of the present invention;

[0082] FIG. 2 is a graph showing the molecular weight distribution of the heparin oligosaccharides with a same degree of polymerization in Example 1 of the present invention;

[0083] FIG. 3 is a total ion spectrum in the UPLC-MS of the heparin decasaccharide obtained in Example 1 of the present invention;

[0084] FIG. 4 is a total ion spectrum (partial) in the UPLC-MS of the heparin decasaccharide obtained in Example 1 of the present invention, and the attribution of corresponding peaks;

[0085] FIG. 5 shows HSQC spectra and the corresponding one-dimensional hydrogen spectra of the heparin octasaccharide (A) and 40% sulfated heparin octasaccharide (B) (H ppm 6.1 to 3.165/C ppm 112 to 52.6);

[0086] FIG. 6 is a graph showing the results of the inhibitory activity of the sulfated heparin oligosaccharides on heparanase;

[0087] FIG. 7 is a graph showing the results of the inhibitory activity of the sulfated heparin oligosaccharide on cell adhesion of HeLa cells;

[0088] FIG. 8 is a graph showing the results of the inhibitory activity of the sulfated heparin oligosaccharide on cell migration of HeLa cells.

DETAILED DESCRIPTION OF THE INVENTION

[0089] The technical solutions of the present invention will now be further illustrated by the following specific embodiments. As can be appreciated by a person skilled in the art, the Examples are given merely to aid understanding of the invention, and should not be considered limitative on the invention.

Example 1. Preparation of Heparin Oligosaccharides

[0090] 24 g of heparin was taken and added with 240 mL of a Tris-HCl buffer solution, which was stirred to dissolve, added with 480 IU of heparinase I, stirred until homogeneous, and underwent an enzymatic reaction at 10 C. for 16 h. After the reaction was complete, the reaction mixture was heated to 95 C. for inactivation for 6 min, and ultrafiltered with a 10 KDa ultrafiltration centrifuge tube. The filtrate was separated with a Bio-Gel P-10 (2.5100 cm) chromatographic column with 0.2 M NH.sub.4HCO.sub.3 as the eluent, 1.75 to 2.15-fold column volume of fractions was collected to obtain a heparin tetrasaccharide mixture, 1.35 to 1.75-fold column volume of fractions was collected to obtain a heparin hexasaccharide mixture, 1.05 to 1.35-fold column volume of fractions was collected to obtain a heparin octasaccharide mixture, 0.85 to 1.05-fold column volume of fractions was collected to obtain a heparin decasaccharide mixture, and 0.75 to 0.85-fold column volume of fractions was collected to obtain a heparin dodecasaccharide mixture. After removal of NH.sub.4HCO.sub.3 through rotary evaporation, the corresponding heparin oligosaccharides were obtained by lyophilization.

[0091] As shown in FIG. 1 which is a graph showing the separation of the heparin oligosaccharides by a Bio-Gel P-10 (2.5100 cm) chromatographic column, it can be seen that after separation by the P10 column, heparin oligosaccharides are separated into different peaks based on different molecular weights, and oligosaccharides with different degrees of polymerization can be obtained by collecting fractions at the tips of the peaks.

[0092] FIG. 2 shows the molecular weight distribution of the obtained heparin oligosaccharides with a same degree of polymerization. It can be seen from the graph that the molecular weight distribution peaks of the oligosaccharides are sharp and symmetrical, indicating that the molecular weight distribution of the oligosaccharides was in narrow ranges, and meanwhile the molecular weight distribution gradually decreases from dodecasaccharide to tetrasaccharide, indicating that the oligosaccharides met the quality requirements.

[0093] The structures of the heparin oligosaccharides were characterized and verified by means of UPLC-MS etc. For example, FIG. 3 is a total ion mass spectrum in the UPLC-MS of the heparin decasaccharide obtained in the present invention, and FIG. 4 is a graph showing the attribution of corresponding peaks in the total ion spectrum of the heparin decasaccharide, wherein in Ux,y,z (specific values of x, y and z are given in the graph), x represents the number of saccharide units in an oligosaccharide chain, y represents the total number of sulfonic groups in an oligosaccharide chain, z represents the total number of acetyl groups in an oligosaccharide chain; and LR represents the linking region. According to the structure attribution in FIG. 4, in the heparin decasaccharides, the oligosaccharides are mostly heparin decasaccharides, with only a small portion being highly sulfated heparin octasaccharides. The above characterization results indicate that the preparation of oligosaccharides is successful and meets the purity requirements.

Example 2. Preparation of 20% to 80% Sulfated Heparin Octasaccharides

[0094] 0.54 g of the starting material of heparin octasaccharide was weighed and transferred to a reaction flask, added with 25 mL of anhydrous DMF, and stirred to dissolve. With stirring, 0.86 g of (CH.sub.3).sub.3N.SO.sub.3 was weighed and gradually added to the above solution, and stirred for 10 min. The reaction flask was capped appropriately, placed in an oil bath at 80 C.n and stirred for 4 h. The reaction was stopped before left to cool to room temperature. The solid was dissolved in 90 mL of purified water, and the pH was adjusted to near neutral with 2 M NaOH. The solution was transfer to a dialysis bag with a molecular weight cut-off of 100 to 500 Da, and after three days of dialysis, the dialysate was detected with BaCl.sub.2 to determine whether a large amount of sulfate radicals are present therein. If not, the pH was first adjusted to neutral with 2 M NaOH, and concentrated to about 10 mL on a rotary evaporator. The mixture was loaded on a P2 column, eluted with purified water, and the eluent was collected with a fraction collector. The absorbance at 232 nm was detected to determine the location of the heparin octasaccharide derivative, the collected solution was detected with BaCl.sub.2 to determine whether a large amount of sulfate radicals are present therein, and the salt-free fraction of the heparin octasaccharide sulfated derivative was collected. The material was concentrated to about 10 mL, loaded on a Dowex cation exchange column, eluted with purified water, the eluent was collected with a fraction collector, and the absorbance at 232 nm was detected to determine the location of the heparin octasaccharide derivative. The fraction comprising the heparin octasaccharide derivative was collected, carefully adjusted to pH 7.0 with 0.1 M high-purity NaOH solution, and concentrated and lyophilized to obtain 20% sulfated heparin octasaccharide.

[0095] For 0.54 g of the starting material of heparin octasaccharide, heparin oligosaccharides with the indicated sulfation degrees can be obtained in a controllable manner by adjusting the feed amount of (CH.sub.3).sub.3N.SO.sub.3 in the starting materials of the reaction and adjusting the reaction temperature and the reaction time as shown in Table 1.

TABLE-US-00001 TABLE 1 Sulfation Reaction Reaction Degree Starting Material Temperature Time 20% sulfation 0.835 g (CH.sub.3).sub.3NSO.sub.3 80 C. 2-6 h 40% sulfation 1.67 g (CH.sub.3).sub.3NSO.sub.3 90 C. 2-6 h 60% sulfation 3.34 g (CH.sub.3).sub.3NSO.sub.3 90 C. 4-8 h 100% sulfation 5.01 g (CH.sub.3).sub.3NSO.sub.3 100 C. 6-10 h

[0096] For example, heparin oligosaccharides with corresponding sulfation degrees can be obtained through the adjustment of the reaction conditions and the feed amount as shown below.

[0097] The amount of (CH.sub.3).sub.3N.SO.sub.3 was adjusted to 1.67 g, the reaction temperature was adjusted to 90 C., the reaction time was adjusted to 4 h, and the remaining procedures were the same, so as to obtain 40% sulfated heparin octasaccharide.

[0098] The amount of (CH.sub.3).sub.3N.SO.sub.3 was adjusted to 3.34 g, the reaction temperature was adjusted to 90 C., the reaction time was adjusted to 6 h, and the remaining procedures were the same, so as to obtain 60% sulfated heparin octasaccharide.

[0099] The amount of (CH.sub.3).sub.3N.SO.sub.3 was adjusted to 5.01 g, the reaction temperature was adjusted to 100 C., the reaction time was adjusted to 8 h, and the remaining procedures were the same, so as to obtain 80% sulfated heparin octasaccharide.

[0100] FIG. 5 shows HSQC spectra and the corresponding one-dimensional hydrogen spectra of the heparin octasaccharide (A) and 40% sulfated heparin octasaccharide (B) (.sub.H ppm 6.1 to 3.165/.sub.C ppm 112 to 52.6). It can be seen from the graph that after the sulfation reaction, the signal peaks of the heparin octasaccharide change significantly, and there are many new signals, indicating the presence of structures different from natural heparin, which is consistent with the expectation of sulfated derivatization.

Example 3. Heparanase Inhibitory Activity of 40% and 60% Sulfated Heparin Octasaccharides

[0101] The heparanase inhibitory activity of sulfated heparin oligosaccharides was determined with the method as described in the reference [Hammond E, Li C P, Ferro V. Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening. Anal. Biochem. 2011; 396:112-116]. Specifically, the experimental procedure were as follows.

[0102] The reaction solution of the test group comprised 40 mM sodium acetate buffer (pH 5.0), 100 mM fondaparinux, and a specific concentration of the sulfated heparin octasaccharide, and in the reaction solution of the control group, the sulfated heparin octasaccharide was replaced with SST0001 control at a same concentration as that of the sulfated heparin octasaccharide. 100 L of the reaction solution of the test group or the control group was added to each well in a 96-well plate, and heparanase was respectively added to a final concentration of 140 pM, to start the reaction. The 96-well plate was sealed with a tape, and incubated at 37 C. for 2 to 24 h. After the reaction was complete, 100 L of a solution containing 1.69 mM WST-1 in 0.1 M NaOH was added to stop the reaction. The 96-well plate was resealed and incubated at 60 C. for 60 min. After cooling to room temperature, the absorbance value at 584 nm was measured. The inhibitory rate of the agent was calculated according to the following method:

Inhibitory rate=(1the absorbance value of a sample/the absorbance value of a control)100%

[0103] The heparanase inhibitory activity of the sulfated heparin oligosaccharides measured according to the above method is shown in FIG. 6. It can be seen from the graph that the half maximal inhibitory concentration (IC50) of 40% sulfated heparin octasaccharide (Hep8-40%) on heparanase was 43 ng/mL, and the half maximal inhibitory concentration (IC50) of 60% sulfated heparin octasaccharide (Hep8-60%) on heparanase was 57 ng/mL.

Example 4. Cell Adhesion Assay of 20% and 60% Sulfated Heparin Octasaccharides

[0104] In this example, the following method was employed to determine the cell adhesion of 20% and 60% sulfated heparin octasaccharides.

[0105] (1) coating the basement membrane: 2 solutions were prepared with sterile double distilled water respectively: 10 g/L of BSA (1%), and 50 mg/L of Matrigel, 1:8 dilution; Matrigel was added to a 96-well culture plate at 50 L/well respectively, and incubated at 4 C. overnight;

[0106] (2) hydrating the basement membrane: the residual liquid was aspirated from the culture plate, 50 L of serum-free medium containing 10 g/L of BSA was add to each well, and allowed to stand at 37 C. for 30 min.

[0107] (3) preparing cells: normally cultured HeLa cells were taken and washed with PBS for three times, and the cells were divided into a control group (no drug but only a corresponding volume of PBS was added) and test groups (heparin and sulfated heparin octasaccharide were added, both at a concentration of 62.5 g/mL), and cultured for 24 h.

[0108] (4) inoculating cells: tumor cells normally cultured or treated with heparin/HS oligosaccharide derivatives in step (3) for 24 h were taken, and washed with PBS for three times before digested with 0.5 mL of pancreatin. The digestion was closely monitored, and after the digestion, 5 mL of the culture medium was added, and the cells were dispersed by thoroughly pipetting to form a single cell suspension. The cells were counted, and the concentration of the cell suspension was adjusted according to the counting result to achieve a cell density of 10.sup.5 cells/mL. The cell suspension was inoculated at 100 L per well to a 96-well culture plate coated with Matrigel, and samples were processed in triplicate;

[0109] (5) culturing cells: the cells were cultured in a carbon dioxide incubator at 37 C. for 1 h, and washed with PBS once after the culture liquid in each well was discarded. 200 L per well of fresh culture medium was then added, and the plated was observed and photographed.

[0110] (6) detecting: 10 L of a CCK-8 dye solution was added to each well, and the plate was incubated in a carbon dioxide incubator at 37 C. for 3 hours, and read with a multifunctional microplate reader. The inhibitory rate was calculated according to the following formula, with the heparin control group as a reference.

Inhibitory rate=(1the absorbance value of the treatment group/the absorbance value of the control group)100%

[0111] According to the above method, the inhibitory rate of heparin on HeLa cell adhesion was 8.3%, the inhibitory rate of 20% sulfated heparin octasaccharide (Hep8-20%) on HeLa cell adhesion was 45.3%, and the inhibitory rate of 60% sulfated heparin octasaccharide (Hep8-60%) on HeLa cell adhesion was 37.9%.

Example 5. Cellular Migration Assay with 40% and 60% Sulfated Heparin Octasaccharides

[0112] In this example, cell migration with 40% and 60% sulfated heparin octasaccharides was determined by the following method.

[0113] (1) before the test, a Transwell chamber was placed in a 24-well plate, 600 L of a DMEM liquid medium was added to the lower chamber, 100 L of a DMEM liquid medium was added to the upper chamber, and the chamber was left in an incubator overnight for further use;

[0114] (2) preparation of cells: HeLa cells in logarithmic growth phase were taken and digested with pancreatin, the cell concentration was counted, and the cells were divided into a control group (no heparin or heparin oligosaccharides but only a corresponding volume of PBS was added) and test groups (heparin and sulfated heparin octasaccharide were added, both at a concentration of 62.5 g/mL). The cells were diluted with a serum-free DMEM medium containing 2.5% BSA and serum-free, 2.5% BSA DMEM medium containing a corresponding heparin oligosaccharide to adjust the cell concentration to 2.510.sup.5 cells/mL.

[0115] (3) 800 L of a culture medium containing 5% FBS was added to the lower chamber of the Transwell chamber.

[0116] (4) 400 L of the cell suspension in step (2) was added to the upper chamber of the Transwell chamber.

[0117] (5) The chamber was incubated in an incubator at 37 C. under 5% CO.sub.2 for 8 hours.

[0118] (6) The liquid in the upper chamber was carefully removed, and the inner surface of the chamber membrane was gently wiped with a cotton swab to remove non-migrating cells. It should be noted that the operation should be gentle so as to avoid damaging the chamber membrane.

[0119] (7) The chamber was transferred to another 24-well plate containing 400 L of cell dye liquor, and the cells were stained at room temperature for 10 minutes.

[0120] (8) The Transwell chamber was gently washed with distilled water for 3 to 5 times, and left at room temperature for natural-air drying.

[0121] (9) The cells were counted under a microscope and photographed.

[0122] (10) The chamber was transferred to another clean 24-well plate, 200 L of a cell lysis solution was added to each well, and the chamber was placed on a shaker.

[0123] (11) After incubated at room temperature for 10 minutes, 100 L of the cell lysis solution was added to a 96-well plate, and the absorbance value was read at 560 nm. With the heparin control group as a reference, the transfer rate and inhibitory rate were calculated according to the following formulae:

Transfer rate=the absorbance value of the treatment group/the absorbance value of the control group100%

Inhibitory rate=[1(the adhesion rate of the treatment group/the adhesion rate of the control group)]100%

[0124] According to the above method, the results of cell adhesion are shown in FIG. 7, showing that that the inhibitory rate of heparin on HeLa cell migration is 12.6%, the inhibitory rate of 40% sulfated heparin octasaccharide (Hep8-40%) on HeLa cell migration is 57.3%, and the inhibitory rate of 60% sulfated heparin octasaccharide (Hep8-60%) on HeLa cell migration is 43.5%.

Example 6. Anti-Tumor Metastasis Activity of 40% and 60% Sulfated Heparin Octasaccharides

[0125] In this example, the anti-tumor metastasis activity of 40% and 60% sulfated heparin octasaccharides was determined according to the following method.

[0126] B16-BL6 mouse melanoma cells (2105) were injected into C57BL/6 mice via the tail vein, and the mice were divided into a control group (no heparin oligosaccharide was added, and PBS was employed as control) and test groups (40% sulfated heparin octasaccharide was added, 200 g for each mouse), 10 mice per group. After 3 weeks, the mice were dissected, the lungs of the mice were fixed in the Bouin's solution, and the numbers of tumors in the lungs of the mice were calculated. We detected the formation of metastatic tumor weekly through an IVIS-200 fluorescence imaging system by employing luciferase-labeled B16-BL6 mouse melanoma cells. Pictures were taken 10 min after intraperitoneal injection of 2.5 mg of fluorescein.

[0127] According to the above method, the results of anti-tumor metastasis activity are shown in FIG. 8. It can be seen from the graph that the inhibitory rate of heparin on tumor metastasis in mice is 17.3%, the inhibitory rate of 40% sulfated heparin octasaccharide (Hep8-40%) on tumor metastasis in mice is 65.1%, and the inhibitory rate of 60% sulfated heparin octasaccharide (Hep8-60%) on tumor metastasis in mice is 56.2%.

[0128] The applicant hereby states that the above-mentioned examples in the present invention are provided by way of illustration of the sulfated heparin oligosaccharide of the present invention, the preparation method and use thereof, but the present invention is not limited to the above-mentioned examples, i.e. it is not intended that the present invention has to rely on the above-mentioned examples for implementation. It should be appreciated by a person skilled in the art that any modification to the present invention, equivalent replacement of materials employed and addition of an adjunct ingredient in the present invention, as well as selection of specific embodiments etc. would fall within the protection and disclosure scope of the present invention.