CN102070725B - Sulfated galactan, and preparation method and application thereof - Google Patents

Abstract

The invention discloses sulfated galactan, which has the molecular weight of 8.5*10<4>. The mass percentage of the sulfate group is between 25 percent and 27 percent. A chemical structure is irregularly formed by a repeated unit A, a repeated unit B, a repeated unit C and a repeated unit D, wherein the mol percentage of the repeated unit A is 10 to 13 percent, the mol percentage of the repeated unit B is 6 to 7 percent, the mol percentage of the repeated unit C is 63 to 65 percent, the mol percentage of the repeated unit D is 17 to 19 percent, and the total value of the mol percentage of the four repeated units is 98 to 100 percent. The sulfated galactan is prepared according to the following steps: firstly, extracting grateloupia filicina by water, and then, carrying out alcohol deposition, freeze drying and separation purification operation on extracted liquid. The sulfated galactan disclosed by the invention can be used for preparing anti-blood-vessel-generation and/or anti-tumor medicine and can be particularly used for preparing anti-S-180-sarcoma and liver cancer medicine.

Description

A kind of galactan sulfate and its preparation method and application

technical field

The present invention relates to a kind of galactan sulfate and its preparation method and application, specifically, it relates to a kind of uniform galactan sulfate separated from the polysaccharide extract of centipede algae and its preparation method and application , belongs to the technical field of natural medicines.

Background technique

Sulfated polysaccharides in seaweed have attracted extensive attention at home and abroad in recent years due to their various biological activities such as anti-oxidation, anti-virus, anti-coagulation and anti-tumor. Centipede algae is a kind of algae of the genus Centipede algae in the family Halmaceae. Centipede algae has been used as a marine traditional Chinese medicine since ancient times. It has the effects of clearing away heat, detoxifying and repelling insects, and is rich in resources. distributed and eaten by the local people. Polysaccharide is one of the main components of centipede algae. The research results in recent years show that it has good biological activities such as anticoagulant and antiviral, showing its potential value of being developed into a drug. Although Chinese patent document ZL 200510023551.0 discloses a polysaccharide extract of Centipede longifolia, its preparation method and its use in the preparation of anti-angiogenesis and/or anti-tumor drugs; and the patent No. ZL200610026830.7 discloses a The invention relates to the use of centipede algal polysaccharide extract in the preparation of anticoagulant, antithrombotic, anti-AIDS and/or antitumor drugs. However, the polysaccharide extract of centipede algae disclosed in the above-mentioned patent documents is a crude polysaccharide composed of multiple components. It is necessary to apply the polysaccharide extract derived from centipede algae to clinical practice to produce an effective, safe and clear action mechanism. For drugs, further in-depth clinical research on the separation, structure confirmation and drug efficacy of the active ingredients in its polysaccharide extract is required.

Contents of the invention

The purpose of the present invention is to provide a kind of homogeneous galactan sulfate isolated from the polysaccharide extract of centipede algae and its preparation method and application, so as to lay the foundation for the wide application of centipede algae polysaccharide extract.

In order to realize the foregoing invention object, the technical scheme that the present invention adopts is as follows:

Galactan sulfate provided by the invention is characterized in that:

The molecular weight is 8.5×10 ⁴ ; the mass percentage of sulfuric acid groups is 25-27%;

The chemical structure is randomly composed of repeating unit A, repeating unit B, repeating unit C and repeating unit D, wherein: the molar percentage of repeating unit A is 10-13%, and the molar percentage of repeating unit B is 6-10%. 7%, the molar percentage of repeating unit C is 63-65%, and the molar percentage of repeating unit D is 17-19%;

The chemical structural formula of the repeating unit A is as follows:

The chemical structural formula of the repeating unit B is as follows:

The chemical structural formula of the repeating unit C is as follows:

The chemical structural formula of the repeating unit D is as follows:

The sum of the mole percentages of the above four repeating units is 98-100%.

The sulfated galactan of the present invention also contains 1,3-linked D-galactose containing pyruvate groups with a molar percentage of 0-2%.

The preparation method of galactan sulfate provided by the invention comprises the following concrete steps:

a) Water extraction: Rinse the freshly collected centipede algae with tap water to remove the surface salinity, chop them up, add distilled water, adjust the pH to 5-7 with glacial acetic acid, and extract at 85-95°C for 0.5-1.5 hours; the residue follows the above process Extract 1 more time; combine the 2 extracts;

b) Alcohol precipitation: add 2 to 4 times the volume of ethanol of the above extract under stirring, let stand, and centrifuge to collect the precipitate;

c) freeze-drying: dissolving the obtained precipitate in water, dialyzing for 24-72 hours, and then freeze-drying;

d) Separation and purification: after dissolving the above lyophilized polysaccharide in a phosphate buffer solution with a pH of 7.2 and a concentration of 20mmol/L, it was chromatographed on a DEAE-Sepharose Fast Flow gel column, firstly with a concentration of 0.5mol/L sodium chloride solution and pH = 7.2, concentration of 20mmol/L phosphate buffer solution to elute 2 times the column volume, and then use concentration of 0.8mol/L sodium chloride solution and pH = 7.2, concentration is 20mmol/L Eluted with phosphate buffer solution, collected by phenol-sulfuric acid method, dialyzed for 24-72 hours, freeze-dried; then dissolved in distilled water, purified with Sepharose CL-6B column, eluted with distilled water, collected by sulfuric acid phenol method, frozen and drying to obtain the galactan sulfate described in the present invention.

The water extraction conditions in step a) are preferably recommended as follows: adjust the pH to 6 with glacial acetic acid, and extract at 90° C. for 1 hour.

The alcohol precipitation condition in step b) is preferably recommended as follows: add 3 times the volume of the above-mentioned extract solution with ethanol with a volume percentage of 95% under stirring.

The dialysis time in step c) is preferably recommended to be 48 hours.

The running water dialysis time in step d) is preferably recommended to be 48 hours.

The present invention also provides the application of the galactan sulfate in the preparation of anti-angiogenesis and/or anti-tumor drugs, especially the application in the preparation of anti-S-180 sarcoma and liver cancer drugs.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The present invention separates and purifies a uniform galactan sulfate from the polysaccharide extract of Centipede Algae for the first time, and conducts drug efficacy research on it, and can prepare a kind of effective, safe and effective The drug with a clear mechanism provides a valuable way, which makes it possible for the centipede algal polysaccharide extract to be widely used.

Description of drawings

Fig. 1 is the high performance gel exclusion chromatogram (HPGPC figure) of the GFP08 sample that embodiment 1 makes.

Fig. 2 is the infrared spectrum of the GFP08 sample that embodiment 1 makes.

Figure 3 is the infrared spectrum of the GFP08 sample prepared in Example 1 after alkali modification.

FIG. 4 is the ¹³ C-NMR spectrum of the GFP08 sample prepared in Example 1.

Figure 5 is an inverted photomicrograph of the human umbilical vein endothelial cell HUVEC lumen under the action of different concentrations of GFP08, wherein: a is the blank control group, b is the 10mg/L GFP08 group, c is the 50mg/L GFP08 group, and d is the 100mg/L GFP08 group.

Figure 6 is a mathematical statistical diagram of the inhibitory effect of different concentrations of GFP08 on the lumen of human umbilical vein endothelial cells HUVEC, in the figure: ** indicates p<0.01 compared with the control group, *** indicates p<0.001 compared with the control group.

Figure 7 is an inverted photomicrograph of human umbilical vein endothelial cells HUVEC under no induction group, 20% FBS induction group, positive drug control group and drug group, wherein: a is no induction group, b is 20% FBS induction group , c is the positive drug control group (20% FBS+10 μM Suramin group), d is the drug group (20% FBS+100 mg.L ^-1 GFP08 group).

Figure 8 is a mathematical statistical diagram of the inhibitory effect of GFP08 on the migration of human umbilical vein endothelial cells HUVEC in terms of OD value. In the figure: FBS1 is the positive drug control group (20% FBS+10μM Suramin group), and FBS2 is the drug group (20% FBS +100 mg.L ^-1 GFP08 group); * indicates p<0.05 compared with the control group, ** indicates p<0.01 compared with the control group.

Figure 9 is a mathematical statistical diagram of the inhibitory effect of GFP08 on the migration of human umbilical vein endothelial cells HUVEC in terms of the number of cell migration. In the figure: FBS1 is the positive drug control group (20% FBS+10μM Suramin group), and FBS2 is the drug group (20 %FBS+100 mg.L ^-1 GFP08 group); ** indicates p<0.01 compared with the control group, *** indicates p<0.001 compared with the control group.

Figure 10 is a graph showing the expression of 100mg/L GFP08 on the TF protein of human umbilical vein endothelial cells HUVEC.

Detailed ways

Below in conjunction with embodiment, the present invention is described in further detail and completeness:

Example 1

1. Preparation of galactan sulfate

Rinse the freshly collected centipede algae with tap water to remove the surface salinity, chop them up, add distilled water, adjust the pH to 6 with glacial acetic acid, and extract at 90°C for 1 hour; extract the residue once more according to the above process; combine the two extracts ;

Add 3 times the volume of the above extract with 95% ethanol under stirring, let it stand, and centrifuge to collect the precipitate;

The resulting precipitate was dissolved in water, dialyzed for 48 hours, and then freeze-dried;

Take 3g of the above freeze-dried polysaccharide and dissolve it in 100mL of phosphate buffer solution with pH=7.2 and concentration of 20mmol/L, and then use DEAE-Sepharose Fast Flow gel chromatography column (2.6cm×60cm) mol/L sodium chloride solution and pH=7.2, the concentration is 20mmol/L phosphate buffer solution to elute 2 times the column volume, and then the concentration is 0.8mol/L sodium chloride solution and pH=7.2, the concentration is 20mmol/L phosphate buffer solution was eluted, collected by phenol-sulfuric acid method, running water dialysis for 48 hours, and freeze-dried to obtain 500mg of the sample; the 500mg sample was dissolved in 5mL of distilled water, and the Sepharose CL-6B column (2.6cm×100cm ) purification, eluted with distilled water, detected by the sulfuric acid phenol method, collected 43 to 55 tubes (5ml per tube), freeze-dried to obtain 250 mg of the galactan sulfate (marked as GFP08) sample of the present invention.

2. Determination of purity and molecular weight

Detected by HPGPC, the detector is a differential detector, the chromatographic column is Shodex KS-805 and KS-804 connected in series, 0.2mol/L sodium chloride aqueous solution is used for elution, dextran with different molecular weights is used as a standard, and AgilentGPC software is used according to The elution time was used to calculate its molecular weight.

The prepared GFP08 sample was detected by HPGPC as a single symmetrical peak (as shown in Figure 1), indicating that the galactan sulfate described in the present invention is a homogeneous polysaccharide with a molecular weight of 8.5×10 ⁴ .

3. Structural identification

1. Sugar composition analysis

The monosaccharide composition of GFP08 sample was determined by reduction hydrolysis method, the content of sulfate group was determined by barium sulfate turbidimetry, the content of pyruvate group was determined by 2,4-dinitrophenylhydrazine method, and the content of D- and L-galactose was determined using chiral The reagent (S)-(+)-isopropanolamine was determined according to the two-step hydrolytic reduction ammoniation method reported by Navarro, D.A. et al. The measurement results are shown in Table 1.

Sugar composition and molecular weight of table 1GFP08

Note: The absolute configurations of 3,6-anhydrogalactose and 6-methylgalactose were determined by ¹³ C NMR.

As can be seen from Table 1: GFP08 is a galactan sulfate, and D-galactose (D-galactose+6-methyl-D-galactose) and L-galactose (L-galactose+L- 3,6 anhydrogalactose) in a ratio close to 1:1, indicating that the polysaccharide has an agar-type structure.

2. Methylation analysis

From the methylation results of GFP08DS (see Table 2), it can be seen that GFP08 is mainly composed of 1,3-linked D-galactose, 2-sulfated D-galactose and 1,4-linked 2,3 Sulfated L-galactose, 3,6 anhydro L-galactose, 6-sulfated L-galactose.

Table 2 Methylation analysis results of GFP08

3. IR analysis

In the infrared spectrum of GFP08 (as shown in Figure 2): there are characteristic absorption peaks of sulfate ester group (1260cm ^-1 ) and 3,6-anhydrogalactose (935cm ^-1 ), and the absorption peak around 842cm ^-1 indicates that the polysaccharide Sulfate group is substituted at position 2, 3 or 4, and there is a shoulder peak at 820cm ^- 1 indicating that there is a 6-sulfate group substitution; after alkali modification (as shown in Figure 3), the shoulder peak at 820cm ^-1 It is much weaker, which indicates that the 6-sulfate group is substituted on the 1,4-linked galactose. After alkali treatment, the 6-position sulfate group is removed and condensed with the 3-position hydroxyl to form 3,6 anhydrogalactose.

4. ¹³ C NMR analysis

In the ¹³ C NMR of GFP08 (shown in Figure 4), there are mainly four anomeric carbon signal peak positions: 104.4, 103.0, 102.1, and 99.2, and there is no absorption peak at a field higher than 99.2, indicating that the polysaccharide does not contain 1, 4 The presence of linked α-L-galactose and 3,6 anhydro α-L-galactose (99.2) further confirms that its structure is agar-type; see Table 3 for the detailed assignment.

Table 3 Assignment of GFP08 ¹³ C NMR signals

Note: D stands for 1,3-linked D-galactose, D2S stands for 2-sulfated 1,3-linked D-galactose, DP stands for pyruvate-containing 1,3-linked D-galactose, D6M stands for 6-position methyl 1, 3-linked D-galactose, L2S3S represents 2, 3-position double sulfated 1, 4-linked L-galactose, L6S represents 6-position sulfated 1, 4-linked L-galactose, LA stands for 3,6-anhydro1,4-linked L-galactose. ^a Due to the low content, the signal is unclear and only partially assigned.

In summary, it can be deduced that the sulfated galactan of the present invention is composed of repeating unit A, repeating unit B, repeating unit C and repeating unit D randomly, wherein: the molar percentage of repeating unit A is 10～ 13%, the molar percentage of repeating unit B is 6-7%, the molar percentage of repeating unit C is 63-65%, the molar percentage of repeating unit D is 17-19%, the four repeating units The sum of mole percentages is 98-100%, and may also contain pyruvate-containing 1,3-linked D-galactose with a mole percentage of 0-2%.

Example 2

Well-grown 7-11 day old S-180 sarcoma tumors were taken, the tumor liquid was made into 1×10 ⁷ /ml cell suspension, and 0.1 ml/mouse was inoculated subcutaneously in the right axilla of mice. 24 hours after inoculation, the cages were randomly divided, and 24 hours later, different concentrations of GFP08 were administered intravenously for 12 consecutive days. The positive drug 5-FU was administered every two days after tumor inoculation. The animals were sacrificed 24 hours after drug withdrawal, the body weight and tumor weight were weighed, and the average tumor weight of each group was calculated. The tumor inhibition rate was calculated according to the following formula and t-test was performed.

Tumor inhibition rate (%)=(average tumor weight of blank control group-average tumor weight of treatment group)/average tumor of blank control group×100%.

Efficacy evaluation criteria: tumor inhibition rate < 40% is invalid; tumor inhibition rate ≥ 40%, and p < 0.05 after statistical processing is effective. See Table 4 for specific data.

The effect of table 4GFP08 on the growth of mouse S-180 sarcoma

As can be seen from Table 4: after the tumor inoculation, the mice were given 25 mg/kg, 50 mg/kg, and 100 mg/kg of the galactan sulfate (GFP08) of the present invention continuously for 12 days, and both the 50 mg/kg and 100 mg/kg groups could Significantly inhibit the growth of S-180 sarcoma, the tumor inhibition rate is 54.7% and 68.9%, the effect is better than the anti-tumor effect disclosed in the Chinese patent application 200610026830.7, indicating that the purified algae polysaccharide improves its anti-tumor effect. The positive control drug 5-FU 25mg/kg was intravenously administered once every two days after tumor inoculation, which significantly inhibited the growth of S-180 sarcoma, and the tumor inhibition rate was 90.5%.

Example 3

90 μl of Bel7402 liver cancer cell liquid was implanted into each well of a 96-well plate, about 6000 cells, and cultured for 24 hours. The GFP08 sample was formulated into three different concentrations of 1.0 mg/ml, 0.1 mg/ml and 0.01 mg/ml, and then 100 μl was added to each well. Three wells were seeded for each concentration. Add 200 μl of culture medium around a circle to prevent edge effects. Cultured for 3 days after dosing. Aspirate all the liquid in the well and wash it once with PBS. (to prevent the sample from reacting with MTT) Then add 20 μl (5 mg/ml) of fresh MTT solution to each well and shake on a shaker for 5 minutes. Continue to incubate at 37°C for 4h after adding the sample. Add 100 μl DMSO to each well and shake for 5 minutes. The absorbance of each well was measured with an enzyme-labeled immunoassay at 570 nm. The inhibition rate was calculated according to the following formula, and the results are shown in Table 5.

Inhibition rate=(1-OD experimental group/OD control group)×100%

Table 5 Effect of GFP08 on the growth of Bel-7402 liver cancer cells

Concentration (mg/ml) 1.0 0.1 0.01 Inhibition rate(%) 98.4 70.0 20.5

It can be seen from Table 5 that GFP08 at 1.0 mg/ml has a significant inhibitory effect on the growth of BEL-7402 liver cancer cells, and the inhibition rate reaches 98.4%.

Example 4

30 μl of liquid ECMatix glue was applied to each well of a 96-well plate and cured at 37°C for 45 minutes. Add 100 μl of HUVEC cell suspension (3×10 ⁴ /well) to each well, add different concentrations of drugs or vehicles, and culture at 37° C., 5% CO ₂ for 8 hours. Images were recorded with an inverted aberration microscope at 250 times, and then the number N of complete lumen formation was calculated. Tube formation rate=(N _{experimental group} -N _{control group} )/N _{control group} ×100%. All experimental data are expressed as mean ± standard deviation, multiple groups were compared using one-way analysis of variance, and SPSS16.0 software was used for statistical analysis.

Figure 5 is an inverted photomicrograph of the human umbilical vein endothelial cell HUVEC lumen under the action of different concentrations of GFP08, wherein: a is the blank control group, b is the 10mg/L GFP08 group, c is the 50mg/L GFP08 group, and d is the 100mg/L GFP08 group.

Figure 6 is a mathematical statistical diagram of the inhibitory effect of different concentrations of GFP08 on the lumen of human umbilical vein endothelial cells HUVEC, in the figure: ** indicates p<0.01 compared with the control group, *** indicates p<0.001 compared with the control group.

Combining Figure 5 and Figure 6, it can be seen that GFP08 can significantly inhibit the tube formation of HUVEC cells in a dose-dependent manner at 10, 50, and 100 mg/L.

Example 5

Immerse the bottom membrane of Millicell in 0.1% gelatin solution, keep the temperature at 37°C for 2 hours, take it out, turn the chamber upside down, and dry it in the air. Take a 24-well plate and set up no induction group, 20% FBS induction group, positive drug control group and drug group respectively. Add 500 μl of M199 medium containing 0.1% BSA to the wells of the no induction group, and add 500 μl of M199 medium containing 20% FBS. Put the dried Millicell into a 24-well plate, add 100 μl of cell suspension (3×10 ⁵ /well) into the small chamber, add solvent or drug in turn, incubate at 37°C, 5% CO ₂ for 24 hours, take it out, and place the bottom of the Millicell Immerse in 4% paraformaldehyde and fix for 2 hours, gently wipe off the cells in the upper chamber with a cotton swab, stain the cells in the lower chamber with 0.1% crystal violet for 30 minutes, and count them under a 100X microscope after drying (take the average value of 5 random fields of view). Then the crystal violet on the stained cells was dissolved in 300 μl of a solution containing 30% acetic acid, and the light absorbance was measured at 600 nm, and the cell mobility=(OD _{experimental group} /OD _{control group} ))×100%. All experimental data are expressed as mean ± standard deviation, multiple groups were compared using one-way analysis of variance, and SPSS16.0 software was used for statistical analysis.

Figure 7 is an inverted photomicrograph of human umbilical vein endothelial cells HUVEC under no induction group, 20% FBS induction group, positive drug control group and drug group, wherein: a is no induction group, b is 20% FBS induction group , c is the positive drug control group (20% FBS+10 μM Suramin group), d is the drug group (20% FBS+100 mg.L ^-1 GFP08 group).

Figure 8 is a mathematical statistical diagram of the inhibitory effect of GFP08 on the migration of human umbilical vein endothelial cells HUVEC in terms of OD value. In the figure: FBS1 is the positive drug control group (20% FBS+10μM Suramin group), and FBS2 is the drug group (20% FBS +100 mg.L ^-1 GFP08 group); * indicates p<0.05 compared with the control group, ** indicates p<0.01 compared with the control group.

Figure 9 is a mathematical statistical diagram of the inhibitory effect of GFP08 on the migration of human umbilical vein endothelial cells HUVEC based on the number of cells. In the figure: FBS1 is the positive drug control group (20% FBS+10μM Suramin group), and FBS2 is the drug group (20% FBS+100 mg.L ^-1 GFP08 group); ** indicates p<0.01 compared with the control group, *** indicates p<0.001 compared with the control group.

Combining Figures 7 to 9, it can be seen that 100mg/L GFP08 can significantly inhibit the migration of HUVEC cells induced by 20% FBS in vitro.

Example 6

According to the following treatment methods, the cells were treated with lysate (50mM Tris (pH7.5), 1mM EDTA, 150mM NaCl, 20mM NaF, 0.5% NP-40, 10% glycerol, 1mM phenylmethanesulfonyl fluoride, 10μg/ ml aprotinin (aprotinin), 10 μg/ml leupeptin (leupeptin), 10 μg/ml pepstatin (pepstatin) A to carry out cracking, after the protein concentration is determined, adjust to equal concentration and equal volume samples. Will prepare Electrophoresis of the sample with SDS-PAGE gel. After transferring the membrane, apply the primary antibody to be detected, and after shaking overnight at 4°C, apply the corresponding secondary antibody. After ECL color development, use X-ray film exposure imaging. In order to reuse protein blot , we use Strip buffer (62.5mM Tris (pH6.7), 20% SDS and 0.1M 2-mercaptoethanol (2-mercaptoethanol) in a water bath at 50°C for 30 minutes to remove the primary antibody that has been applied, and then apply a new one primary antibody.

Figure 10 is the expression diagram of 100mg/L GFP08 on the TF protein of HUVEC. It can be seen from Figure 10 that the anti-tumor and anti-angiogenesis effects of GFP08 may be related to the TF signaling pathway.

In addition, it should be noted that the galactan sulfate of the present invention can be prepared into various pharmaceutical preparations with pharmaceutically acceptable carriers. The "pharmaceutically acceptable carrier" should not destroy the pharmaceutical activity of galactan sulfate, and its effective dosage, that is, the dosage when it can function as a drug carrier, should be non-toxic to the human body, including but not limited to: ion exchange materials, Alumina, aluminum stearate, lecithin, self-emulsifying drug delivery system.

Other pharmaceutically acceptable excipients such as fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as cross-linked sodium carboxymethyl starch, cross-linked carboxymethyl starch Sodium methylcellulose, low-substituted hydroxypropylcellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents , solvents, solubilizers and colorants, etc. can also be added to the pharmaceutical preparation prepared from the galactan sulfate of the present invention.

In particular, the galactan sulfate of the present invention can be prepared as an intravenous administration preparation, and of course, it can also be prepared for administration by enteral or parenteral route, aerosol inhalation or implant accumulation or acupuncture and other methods. pharmaceutical preparations.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the invention can be Modifications or equivalent replacements without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the claims of the present invention.

Claims (10)

1. galactan sulfate is characterized in that:

Molecular weight is 8.5 * 10 ⁴The quality percentage composition of sulfate group is 25～27%;

Chemical structure is by repeating unit A, repeating unit B, repeating unit C and repeating unit D is random forms; Wherein: the molar content of repeating unit A is 10～13%; The molar content of repeating unit B is 6～7%; The molar content of repeating unit C is 63～65%, and the molar content of repeating unit D is 17～19%;

The chemical structural formula of repeating unit A is following:

The chemical structural formula of repeating unit B is following:

The chemical structural formula of repeating unit C is following:

The chemical structural formula of repeating unit D is following:

The summation of the molar content of above-mentioned four kinds of repeating units is 98～100%.

2. galactan sulfate according to claim 1 is characterized in that: also contain molar content in the described galactan sulfate and be 0～2% the D-semi-lactosi that 1,3 of acetone acidic group connects that contains.

3. the preparation method of the described galactan sulfate of claim 1 is characterized in that, comprises following concrete operations step:

A) water extraction: shred after the Grateloupia filicina (Wulf.) of fresh collection rinsed out salt surfactant with tap water, add zero(ppm) water, transfer to pH=5～7, extracted 0.5～1.5 hour at 85～95 ℃ with Glacial acetic acid min. 99.5; Residue extracts 1 time by said process again; Merge 2 times extracting solution;

B) alcohol precipitation: under agitation add 2～4 times of volume of ethanol of said extracted liquid, leave standstill centrifugal collecting precipitation;

C) freeze-drying: gained is precipitated water-soluble, dialysed lyophilize then 24～72 hours;

D) separation and purification: after above-mentioned freeze dried polysaccharide used pH=7.2, the concentration phosphate buffer soln dissolving as 20mmol/L; With DEAE-Sepharose Fast Flow gel chromatography column chromatography; Sodium chloride solution and the pH=7.2, the concentration that earlier with concentration are 0.5mol/L are 2 times of column volumes of phosphate buffer soln wash-out of 20mmol/L; Use concentration to be the sodium chloride solution of 0.8mol/L and pH=7.2, concentration phosphate buffer soln wash-out again as 20mmol/L; The phenolsulfuric acid method detects collects, flowing water dialysis 24～72 hours, lyophilize; Use dissolved in distilled water again, with Sepharose CL-6B column purification, with the zero(ppm) water wash-out, the sulfuric acid phynol method detects to be collected, and lyophilize promptly gets described galactan sulfate.

4. the preparation method of galactan sulfate according to claim 3 is characterized in that, the water extraction condition in the step a) is: transfer to pH=6 with Glacial acetic acid min. 99.5, extracted 1 hour at 90 ℃.

5. the preparation method of galactan sulfate according to claim 3 is characterized in that, the alcohol precipitation condition in the step b) is: the volumn concentration that under agitation adds 3 times of volumes of said extracted liquid is 95% ethanol.

6. the preparation method of galactan sulfate according to claim 3 is characterized in that, the dialysis time in the step c) is 48 hours.

7. the preparation method of galactan sulfate according to claim 3 is characterized in that, the flowing water dialysis time in the step d) is 48 hours.

8. the application of the described galactan sulfate of claim 1 in the preparation angiogenesis inhibitor.

9. the application of the described galactan sulfate of claim 1 in the preparation antitumor drug.

10. the application of the described galactan sulfate of claim 1 in anti-S-180 sarcoma of preparation or liver-cancer medicine.

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